Objective@#To prepare monoclonal antibodies against pneumoniaserotype 33F polysaccharides (Pn33Fps) and hepatitis B virus (HBV) surface proteins (HBs) by using the conjugate of Pn33Fps and HBs as antigen.@*Methods@#The conjugate of Pn33Fps and HBs was used as antigen to immunize mice with different immune doses, different immune procedures and different immune sites. Mousespleencells with higher antibody level after immunization were isolated and fused with SP2/0 myeloma cells. The hybridomacells were screened specifically with Pn33Fps or HBs to prepare corresponding monoclonal antibodies.@*Results@#Serumantibodies against Pn33Fps and HBs were induced by immunizing mice with the conjugate. Monoclonal cell lines capable of continuously expressing antibodies against Pn33Fps or HBs were obtained. It has been proved that the recovery rates of samples of Pn33Fps and HBs prepared in three batches tested with ascitesmonoclonal antibodies prepared by these two monoclonal cell lines were between 95% and 105%.@*Conclusions@#Monoclonal antibodies against Pn33Fps and HBs could be prepared simultaneously by immunizing mice with the conjugate of Pn33Fps and HBs and used for the quantitative detection of Pn33Fps and HBs.