A pyridoxine monoclonal antibody hybridoma cell strain and an application thereof. The invention belongs to the field of food safety immunodetection. In the invention, a pyridoxine complete antigen isprepared and is then mixed with a Freund's adjuvant equivalently until complete emulsification; then a BALB/c mouse is immunized through back subcutaneous injection to obtain a pyridoxine pyridoxinehybridoma cell strain SS0708 that is preserved in China General Microbiological Culture Collection Center and September 5th, 2017 and is assigned the accession number of CGMCC No.14687. The monoclonalantibody secreted by the cell strain has excellent specificity and detection sensitivity (IC50 being 250 ng/mL) to the pyridoxine. The product can be used for establishing an immunodetection method for the content of the pyridoxine in infant foods, dairy products, special medical purposed foods and the like, and has practical application value.