Objective To detect traces of microRNAs (miRNAs) in plasma and assess the expression stability of two common reference genes by stem-loop and poly A polymerase (PAP) real-time quantitative polymerase chain reaction (PCR) method, as miRNAs are the new bio-markers of tumor diagnosis and molecular targeted therapy, and its quantitative research is very important. Methods We extracted miRNAs from plasma of adult Sprague Dawley (SD) rats’ plasma, and detected the expressions of rno-miR-200b-3p and rno-miR-126-3p with stem-loop and PAP real-time PCR quantitative method, with rno-miR-103a-3p and U6 as internal controls. All the results were evaluated by 2△Ct method. Results Compared with PAP method, the stem-loop method reduced Ct value by 2-4 cycles and improved sensitivity by 10 times. In PAP method, the melting curve showed two peaks, a main peak and a small non-specific peak. Yet the melting curve of stem-loop method demonstrated a single specific peak. Furthermore, we validated the stability of internal references in the two real time PCR methods. U6 presented a more stable Ct value than rno-miR-103 in adult SD rats’ plasma samples. Conclusions Stem-loop real-time PCR is recommended as a major way to detect some samples with a low concentration of miRNAs, owing to its high accuracy and sensitivity. However, if a large number of tissue samples is going to be detected, PAP real-time PCR is more suitable and convenient than stem-loop method. U6 is more stable and repeatable than rno-miR-103a-3p as the reference gene to evaluate the semi-quantitative consequence of miRNAs.