Background: Metagenomic next-generation sequence (mNGS) is an emerging powerful pan-pathogen test for the diagnosis of infectious diseases. However, the application of mNGS in acquired immunodeficiency syndrome with opportunistic infections is limited to clinical cases reports and central nervous system infection.In this study, we evaluated the diagnostic value of mNGS in acquired immunodeficiency syndrome(AIDS) with opportunistic infections(OIs).Methods: From January 2018 to February 2021,86 cases were enrolled in this retrospective analysis. All patients underwent mNGS. Clinical data were recorded.Result: In the present study, mNGS identified 76 of 86 infection cases (88.37%).Human betaherpesvirus 5 (CMV) (40.70%), Human gammaherpesvirus 4 (EBV) (40.70%),pneumocystis (31.40%) were the most common pathogens detected. The sensitivity of mNGS (88.37%,76/86) was higher than that of culture (22.10%, 19/86),smear(7%,6/86) and PCR(46.51%,40/86).In the detection of viruses such as (CMV and EBV), the consistency between PCR and mNGS of CMV and EBV was 100%,73.33% respectively. All PCP cases were detected by mNGS. The consistency in detection of talaromyces between culture and mNGS was 75%.mNGS is superior to the common methods such as culture and smear in the detection of mycobacterium tuberculosis and non-mycobacterium tuberculosis. The mNGS findings led to changes in treatment strategies in 47/86 (54.65%) cases. Compared with the patients’treatment before mNGS, patients had lower rate of broad-spectrum antibiotic drugs use during clinical treatment after mNGS 78/86(90.70%) vs 34/86(39.53%)（P<0.0001). Conclusion: mNGS showed a satisfying diagnostic performance in acquired immunodeficiency syndrome with opportunistic infections. mNGS may lead to a more precise antimicrobial treatment and reduced the use of antibiotic medicine.