A method has been established for the stability evaluation of sulfanilamides and quinolones mixed standard solution in methanol. Each standard substance in the mixed standard solution was accurately determined by liquid chromatography tandem mass spectrometry（LC/MS/MS） in different storage conditions and different storage time. The results showed that the mixed standard solution demonstrated excellent homogeneity, with the F-value of each component falling below the theoretical critical value at the 95% confidence level. The solution rate of sulfadiazine, sulfaquinoxoline, norfloxacin and fleroxacin decreased by more than 10% at 40 ℃. Meanwhile, a mixed standard solution, with each component having a concentration of 10.0 μg/mL, maintained satisfactory stability for up to four months when stored in darkness at 4 ℃. After more than 4 months, with the extension of storage time, the degradation of sulfadiazine, sulfamethazine, sulfaquinoxoline, norfloxacin and fleroxacin was more obvious, and the contents of other antibiotics showed a gradually increasing trend. This mixed standard solution has been found suitable for use in residue detection for food and environmental applications, assisting in the identification and evaluation of related analytical methods, with a validated effectiveness lasting for four months.