Objective To evaluate the clinical application of a new mycoplasma pneumoniae DNA Diagnostic Kit (refer to as concentrate One-step method hereafter). Methods A total of 216 clinical specimens were detected by concentrated one-step method and another kind of fluorescence diagnostic kit using boiling method. The results were analyzed and compared. Simultaneously, the gold standard culture test results of these 216 specimens were gathered and compared with the results from the concentrated one-step and boiling method kits. Results The two kits showed excellent agreement The Positive consistency was 98.36%, the negative consistency was 96.80% and the total consistency was 97.68%. 5 cases of discrepant samples were sequenced review, and then the results were performed with the Kappa consistency analysis. The value of Kappa=1.000, showed good consistency between the two methods. In addition, compared with the gold standard culture test results, the two reagents both showed good accuracy. Conclusion The concentrated one-step method showed good consistency compared with the conventional boiling method. The operation is simple and quick and can avoid laboratory contaminations which occur often during routine DNA extraction process. The PCR detection system uses an internal positive control (internal control) to avoid a false negative result. With a simple procedure and precise results, this new diagnostic kit is suitable for routine clinical lab use.