Abstract: Objective To evaluate the clinical application of simultaneous amplification and testing method for detection the RNA of mycoplasma pneumoniae (MP) in diagnosis of MP infection in pediatric patients. Methods During October, 2016 to July, 2017, Throat swab/sputum and serum specimens were collected from children with acute respiratory infections in inpatient departments. Among them, throat swab/sputum specimens were used to detect MP-DNA and MP-RNA, while serum specimens were used to detect MP-Ab. According to the children's course, Group A(disease course ≤ 10 d) and Group B (disease course>10 d) were divided, Based on the results of MP-Ab and MP-DNA detection, the difference of the positive rates of MP-RNA between each group was compared, while the sensitivity, specificity, positive predictive value and negative predictive value of MP-RNA were evaluated. Data were analyzed using SPSS 21.0. Results Compared with the positive rate of MP-DNA, MP-Ab and MP-RNA, there was no significant difference among them. In Group A, the positive rate of MP-RNA (37.8%) was higher than MP-Ab (22.9%), and in Group B, the positive rate of MP-RNA (10.6%) was lower than MP-Ab (23.1%). The difference between the two groups was statistically significant (P=0.003, P=0.001, respectively). Compared with the results of MP-DNA, there was no significant difference in the positive rate of MP-RNA between the two groups. Compared with MP-Ab and MP-DNA, the sensitivity of MP-RNA was 0.63/0.79, the specificity was 0.91/0.96, the positive predictive value was 0.67/0.85, and the negative predictive value was 0.89/0.94. In group A, the sensitivity and specificity of MP-RNA were 0.94/0.88 and 0.79/0.90, respectively. Conclusion Simultaneous amplification and testing Method for detection the RNA of MP has high sensitivity and specificity, and this method for early diagnosis of MP infection had better clinical diagnostic efficiency. It is a new method for the early diagnosis of MP infection.