Pneumocystis jirovecii is a prototypical opportunistic pathogen, causing an asymptomatic or mild infection in normal hosts and fulminating pneumonia (Pneumocystis pneumonia, PCP) in immunocompromised hosts. PCP is a leading cause of morbidity and mortality in immunocompromised patients such as AIDS patients. Microscopic detection of cysts and trophic forms of P. jirovecii in respiratory secretions is simple and useful but may underestimate the P. jirovecii infection. Conventional polymerase chain reaction (PCR) and nested PCR increase the sensitivity and specificity to identify PCP and provide an approach to discriminate PCP from pulmonary P. jirovecii colonization, but the targeted genes and cut-off value from quantitative real-time PCR remain to be determined. Serum (1-3)-β-D-glucan level and the specific serum antibody titer are ancillary indicators for PCP diagnosis. The successful cultivation of P. jirovecii in vitro is an important progress for PCP research. The diagnosis of PCP relies on the combination of these laboratory examinations as well as the clinical presentations.