To understand the effects of different caprylic acid-ammonium sulfate concentration and centrifugal force on purification of HRP-labeled monoclonal antibody against tetrodotoxin (TTX) and to optimize the purification protocol for the antibody in order to provide the technical basis for TTX detection by immunoassay. Methods Under different centrifugal force and concentration of caprylic acid-ammonium sulfate, the antibody was purified and linked with the horseradish peroxidase enzyme (HRP), and the biochemical properties and parameters of HRP-labeled antibody were determined with indirect enzyme-linked immuno sorbent assay. Results The purity of antibody and HRP-labeled antibody obtained by different methods were different as well as other parameters and bioactivities. The best experiment conditions for high productivity and bioactivity of the monoclonal antibody were ascites diluent:lipoic acid (1000∶V/V) and 10000r/min centrifugation.Conclusion The purification protocol was optimized for the dilution and centrifugal conditions based on the productivity, purity and bioactivity.