Abstract: Objective To obtain hybridoma cell lines which can secrete high-titer monoclonal antibodies against human lung squamous cell carcinoma YTMLC-90 cell line. Methods Balb/c mice were immunized by a human lung squamous cell carcinoma YTMLC-90 cell line. And the immunized splenocytes were fused separately with two murine myeloma cell lines SP2/0 and NS-1 using hybridoma technique. And then, positive hybridomas were selected to cloned culture and subcloned by limited dilution. The titers of McAbs from cell culture supernatant and ascites were assayed by ELISA. The isotype of the McAbs were also identified by ELISA and the average number of chromosome of the hybridoma cell lines were counted by a general method after stained with Gimesa. McAb's cross-reaction with different cells and tissues were detected by ABC immunohistochemistry technique. Results Stable hybridomas S2D1, N2D1, N3C5 were constructed. The titers of these 3 McAbs from cell culture supernatant and ascites were 1:512,1:284,1:1024 and 1:105,1:104,1:104 respectively. The isotypes were IgG1, IgG2a and IgG3. The numbers of chromosome were 90~110. No cross-reaction with other cell lines and tissues. Conclusion Specific and high titer antibodies against human lung cancer were successfully prepared.