Objective The ECG lead II characteristics of Beagle dog, rhesus monkey, Japanese white rabbit and tree shrew were analyzed and summarized to provide reference for drug safety evaluation studies. Methods The ECG lead II of healthy adult Beagle dog, rhesus monkey, Japanese white rabbit and tree shrew were recorded to determine the interval of P, PR, QT (QTc), the QRS waves and amplitude of P, R, T waves and the ST shift. Results and Conclusion All the animals had sinus rhythm. All the four species of animals had similar ECG pattern with no particular specific changes, but had some differences of the QRS wave group and T wave. The amplitude of P and T waves in Japanese white rabbit was smaller, and the heart rate of tree shrew was faster than that of the other species of animals.The ECG lead Ⅱ database of the Beagle dog, rhesus monkey, Japanese white rabbit and tree shrew is established.