Objective: To investigate the pathogenic characteristics and drug resistance of nosocomial infection in intensive care unit (ICU). Methods: Nosocomial infection patients who were treated in ICU of Lujiang County People’s Hospital from January 2016 to December 2018 were selected. Pathogen sample culture and drug sensitivity results of these patients were collected from the Laboratory Department. The data were retrospectively investigated and analyzed to compare the changes of pathogens and drug resistance in different periods. Results: In 2016, 2017 and 2018, a total of 238 pathogens were detected, and the main strains were Gram-negative bacteria; the top 3 of 2016 were Acinetobacter, Klebsiella, and Pseudomonas; the top 3 of 2017 and 2018 were Klebsiella, Acinetobacter, and Pseudomonas. There were significant differences in the types and distribution of strains in different years (P<0.05). The resistance of Klebsiella pneumoniae and Pseudomonas aeruginosa to amikacin increased year by year, with statistically significant difference (P<0.05), and the proportion of multiple drug resistant organisms increased. Conclusion: The main pathogens of nosocomial infection in ICU were Gram-negative bacteria. The distribution of bacteria has changed. Drug resistance is on the rise; therefore, strict management of antibiotics should be further strengthened.