Objective The rat ECG acquisition bases on various physiological signal acquisition systems at present. The users can not modify the analysis algorithm inside and these systems with low sampling frequencies are not useful. This article introduces a high-accuracy rat ECG acquisition system which realizes real-time detection of RR intervals. Methods First, we designed an ECG acquisition circuit based on AD620. USB-6008 was used to realize A/D conversion. Then the real-time detection of RR intervals was realized by Labview combined with Matlab by means of template matching method. Finally, to checkout the accuracy of the real-time detection of RR intervals, ECG signals of 10 randomly selected rats were analyzed offline compared with the threshold method. Results The detection rate of the threshold method and the template matching method were 96.62% and 99.16%, respectively. Conclusions High-accuracy rat ECG acquisition system can be realized by means of software combined with hardware. The real-time detection of RR intervals realized by Labview combined with Matlab by means of template matching method is accurate and provides the basis of the rat ECG research.