The aim of the present study is to re-evaluate the clinical application of two-times serologic immunoglobulin M (IgM) tests using microparticle agglutination assay (MAA), an enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) assay in diagnosing Mycoplasma pneumoniae (MP) infection. A retrospective analysis of 62 children with MP pneumonia during a recent epidemic (2019–2020) was conducted. The MAA and ELISA immunoglobulin M (IgM) and IgG measurements were conducted twice at admission and around discharge, and MP PCR once at presentation. Diagnostic rates in each test were calculated at presentation and at discharge. The seroconverters were 39% (24/62) of patients tested by MAA and 29% (18/62) by ELISA. At presentation, the diagnostic positive rates of MAA, ELISA, and PCR tests were 61%, 71%, and 52%, respectively. After the second examination, the rates were 100% in both serologic tests. There were positive correlations between the titers of MAA and the IgM values of ELISA. The single serologic IgM or PCR tests had limitations to select patients infected with MP in the early stage. The short-term, paired IgM serologic tests during hospitalization can reduce patient-selection bias in MP infection studies.