ABSTRACTPCR can be a supplement toTreponemaserological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield ofTreponema pallidumsubsp.pallidumDNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detectT. pallidumDNA by targeting thepolAgene. Simulation media with 106to 100treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. ThepolAassay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of thepolAassay reached 1 × 102treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield ofT. pallidumDNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs.IMPORTANCESyphilis is a sexually transmitted disease caused byT. pallidumthat can spread into the blood.T. pallidumDNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to bloodT. pallidumDNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations ofT. pallidumDNA was improved, and the low sensitivity of blood-basedT. pallidumPCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring bloodT. pallidumDNA.