Objective Evaluate the clinical value of rapid identification of pathogenic organisms positive for blood culture by MALDI-TOF mass spectrometry in combination with two pretreatment methods based on serum-separating gel tubes. Methods A total of 302 positive blood culturing bottles were collected from May to November 2020 in the First Affiliated Hospital of Nanjing Medical University, rapid identification of pathogenic organisms positive for blood culture was used by MALDI-TOF MS after pretreatment with Separation gel-Saponin method and Separation gel- precipitation method respectively，the results were compared with those of culture identification. Results Separation gel-Saponin method could compete the identification within 45 min, and Separation gel- precipitation method within 20min, respectively. The total accuracy rates of MALDI-TOF MS with both methods were 86.81% (250/288) in 288 cases of single bacterial infection specimens, Both methods identified only one of the bacteria and only 1 case of Separation gel-Saponin method was consistent with the culture identification result in 14 cases of multiple bacterial infections. Conclusion The two pretreatment methods of positive blood culture bottles combined with MALDI-TOF MS could identify the pathogens of blood culture directly, separation gel- precipitation method is simple and can be used as a routine pretreatment method for rapid identification of blood culture in our laboratory.