Background: This study aimed to find the linker with minimal impact among chains to fight against the structure and function of cervical cancer (CC) single-chain antibody. Materials and Methods: The original variable region of heavy chain (VH) and variable region of light chain (VL), and the single-chain antibody with linkers of different lengths (n = 1~8) were modeling by homologous modeling, while the peptide chain structure of (Gly₄Ser)_n was utilized by the linkers. Comparison of the similarity of original VH/VL and VHn/VLn was carried out by applying the algorithm of spatial hierarchical alignment based on the spherical coordinates. The fore and aft distance and diffusion radius of alpha (α) were also calculated. The stability of antibody with different linker length was then compared. ELISA method was adopted to evaluate the immunological activity of single-chain antibody with different linkers. MTT assay was used to analyze the inhibition effect of ScFv-n on CC cells. Results: When n = 4, the structures were the most similar between ScFv and the original VH/VL. When n = 3, the influence of adding connecting peptide on the stability of single-chain antibody was the least. The result of ELISA and MTT methods indicated that when n = 3, single-chain antibody gained the highest activity. Conclusion: The optimum length of linker of anti-human CC single-chain antibody was n = 3 from the point of mathematical modeling and biology experiments. This study provided new ideas for the design and constructions of single-chain antibody, and theoretical basis for the treatment of CC.