To determine the neutralization activity and protective efficacy at the cellular level of all recombinant antibodies, we carried out neutralization tests with various SARS-CoV-2 pseudovirus subvariants, such as Wuhan, Beta, Delta, BA.2, BA.5, and XBB. The half-maximum inhibitory concentration (IC₅₀) values were calculated, and the one-way ANOVA was conducted to assess whether there was significant difference among the antibodies to the different subvariants. The one-way ANOVA results indicated significant difference with the p < 0.0001. The recombinant trispecific antibody exhibited superior neutralization activity and broad protective efficacy compared to other bsAbs and native monoclonal antibodies (p < 0.0001), with IC₅₀ values ranging from 5 to 27 ng/mL for different subvariants, which are significantly lower than those of other recombinant antibodies (Figure 3a–f). Among bispecific antibodies (bsAbs), F-S309 + S2P6 demonstrates superior and broader efficacy compared to others against various SARS-CoV-2 subvariants (p < 0.0001), with IC₅₀ values of 8, 14, 43, 32, 85, and 23 ng/mL, respectively (Figure 4a–f). The bsAbs F-S2P6 + S309, F-h11B111 + S309, and F-S309 + h11B1 exhibit a broader protective spectrum against diverse subvariants. The IC₅₀ of the bsAb F-S2P6 + S309 is 10, 27, 50, 104, 112, and 32 ng/mL against different subvariants (Figure 4a–f). Meanwhile, the IC₅₀ of the bsAb F-h11B11 + S309 is 19, 73, 30, 130, 150, and 73 ng/mL, respectively (Figure 4a–f). And the F-S309 + h11B11 shows a similar trend as the previous bsAbs, with IC₅₀ values of 15, 41, 20, 137, 116, and 64 ng/mL (Figure 4a–f). However, they do not present lower IC₅₀ values compared to F-S309 + S2P6. The bsAbs F-h11B11 + S2P6 and F-S2P6 + h11B11 do not exhibit a similar tendency to other bsAbs (Figure 5). Although F-S2P6 + h11B11 possesses a good neutralization activity against Wuhan and Beta subvariants with IC₅₀ values of 43 and 60 ng/mL (Figure 4a,b), its efficacy is not equivalent against the Delta, BA.1, BA.5, and XBB, which are classified as variants of concern (VOC), with the IC50 values of 333, 358, 327, and 591 ng/mL, respectively. The bispecific antibody F-h11B11 + S2P6 displays a distinctive characteristic in its neutralization profile, with the weakest activity observed against the XBB variant, reflected by an IC₅₀ value of 684 ng/mL (Figure 4f). In contrast, the IC₅₀ values for its neutralizing activity against the other variants are 283, 283, 374, 409, and 372 ng/mL, respectively (Figure 4a–e). Generally speaking, the bsAb F-h11B11 + S2P6 demonstrates the least ideal neutralization activity among the evaluated bsAbs since its IC₅₀ values against all subvariants are relatively higher than those of all other bsAbs. Compared to native monoclonal antibodies S2P6 and h11B11, all these bsAbs display better neutralization activity with lower IC₅₀ values (p < 0.0001), indicating a superior protective efficiency compared to native monoclonal antibodies (Figure 5). S309 exhibits a different scene. The bsAb F-S2P6 + S309, F-h11B111 + S309, F-S309 + S2P6 and F-S309 + h11B11 show a better neutralization activity than S309 against all subvariants with much lower IC₅₀ values. Meanwhile, the bsAb F-S2P6 + h11B11 merely exhibits lower IC₅₀ values against the Beta and BA.5 subvariants in comparison to S309. The bsAb F-h11B11 + S2P6 possesses a superior neutralization activity with lower IC₅₀ values exclusively against the BA.5 subvariant. Additionally, in contrast to the original monoclonal antibody S309, F-h11B11 + S2P6 shows an inferior neutralization activity against other subvariants with significantly higher IC₅₀ values.