Abstract: Objective To explore the distribution of pathogenic microorganisms and the characteristics of multiple infections in AIDS patients with opportunistic infections, and provide the evidence for the early clinical prevention and reasonable drug use. Methods Pathogenic microorganism detection was conducted by using the blood, cerebrospinal fluid and sputum samples collected from hospitalized AIDS patients. Results Among 3 819 hospitalized AIDS patients from 2012 to 2015 in Yunnan Provincial Hospital of Infectious Disease, 56 kinds of pathogenic microorganisms, including fungus, gram-negative bacterium, gram-positive bacterium, virus, spirochete, parasite, were detected in 3 222 AIDS patients, 9 kinds of fungi were detected in 564 patients (17.50%), 23 kinds of gram-negative bacteria in 570 patients (17.69%), 17 kinds of gram-positive bacteria were detected in 135 patients (4.19%), 5 kinds of virus were detected in 1 398 patients (43.39%), Treponema pallidum was detected in 322 patients (9.99%) and Toxoplasma gondii was detected in 233 patients (7.23%). The major fungus was Penicillium marneffei (8.44%), followed by Cryptococcus neoformans and Candida albicans. The major gram negative bacterium was Mycobacterium (10.40%). The major gram positive coccus was Staphylococcus aureus (1.40%). The major virus was hepatitis C virus (28.43%). In clinical samples, the detection rate was highest in blood samples (69.03%), the detection rate in sputum samples was 18.53%, and 8.68% in cerebrospinal fluid samples. Pathogenic microorganisms were detected in 613 abacteria blood, cerebrospinal fluid and bone marrow samples from AIDS patients, meanwhile Mycobacterium tuberculosis was detected in 94 samples. Opportunistic multiple infections with hepatitis C virus, E. coli, Ttreponema pallidum, Mycobacterium tuberculosis and Toxoplasma gondii were observed. In multiple infections with 2 pathogenic microorganisms, hepatitis C virus was the most common one, followed by Mycobacterium. Conclusion The species of pathogenic microorganisms causing opportunistic infections in AIDS patients in Yunnan is complex, hepatitis C virus is most common, followed by Mycobacteria, Treponema pallidum, Penicillium marneffei. It is necessary to pay more attention to multiple infection of hepatitis C virus, Mycobacterium tuberculosis, Treponema pallidum and fungus.