Streptococcus pneumoniae is related to the prognosis of infected patients. If the strain can be identified to the serotype level, it will play a key role in formulating measures to prevent Streptococcus pneumoniae infection. Therefore, this study focuses on analyzing the significance of serotype detection of Streptococcus pneumoniae under the guidance of MALDI-TOF MS technology, and provides a reference for rapid clinical typing of Streptococcus pneumoniae. A total of 500 patients were selected for treatment in our hospital from April 2019 to April 2020. The main types of diseases were patients with sinusitis, pneumonia, bacteremia, and acute otitis media. A total of 500 cases were collected, and specimens of Streptococcus pneumoniae were collected. Observe the distribution of MALDI-TOF MS mass spectrometry scores; the composition of the serotype distribution of Streptococcus pneumoniae isolates; the molecular typing of Streptococcus pneumoniae isolates; the relationship between the clinical characteristics of 500 patients and the distribution of serotypes 14, 19F; the distribution of serotypes 14, 19F and Analysis of the relationship between patient prognosis. Capsular swelling, latex agglutination test, MALDI-TOF MS detection of serotype distribution of serotype Streptococcus pneumoniae isolates, serotypes 6B, 14, 19A, 19F, 23F accounted for a large proportion, of which the highest serotype was mainly 14. 19F; The number of serotypes detected by MALDI-TOF MS was the highest, reaching 489 (97.80%), the number of serotypes detected by capsular swelling was 308 (61.60%), and the number of serotypes detected by latex agglutination test was 313 (62.40%). The distribution of serotypes detected by MALDI-TOF MS was significantly higher than the other two detection techniques (P < 0.05), and there was no statistical difference between the two tests of capsular swelling and latex agglutination (P > 0.05). The distribution rates of serotypes 14 and 19F were compared in different ages, disease types, drugs, etc., and there were statistical differences (P < 0.05), but there was no statistical difference in gender (P > 0.05). The prognosis of 500 patients, of which 487 cases improved (serotype 14: 98 strains; serotype 19F: 100 strains), 13 died (serotype 14: 12 strains; serotype 19F: 12 strains), serotypes 14, 19F There is a significant correlation between the distribution and the prognosis of patients (P < 0.05). MALDI-TOF MS technology can quickly, accurately and simply identify Streptococcus pneumoniae, with high sensitivity and specificity, and can save time for clinical diagnosis of Streptococcus pneumoniae infection. The laboratory can use MALDI-TOF MS to collect protein profiles of known typed strains and construct a corresponding typing mass spectrum library, and perform principal component cluster analysis on the spectrum to be tested and the library, which can quickly type Streptococcus pneumoniae.