To investigate the feasibility of echocardiography-guided closed-chest repeated intraventricular blood sampling in mice, and to clarify the maximum blood volume that can be collected by this method, and whether the method can be used for long-term repeated blood collection in mice. Twenty-four male C57BL/6J mice (10-14 weeks old) were divided into the terminal experiment group (4, for investigating the maximum blood amount that could be sampled at one time), the repeated 0.5 ml blood collection group (10, sampling 0.5 ml whole blood each time, once every two days for consecutive 4 weeks), and the repeated 0.75 ml blood collection group (10, sampling 0.75 ml whole blood each time, once every two days for consecutive 4 weeks). High-frequency echocardiography was used to display the largest section of the left ventricle, guiding the insulin syringe needle through the thorax into the left ventricle for blood collection. In the repeated 0.5 ml blood collection group, echocardiography was used to detect the cardiac structure and function before blood collection, three minutes after blood collection, and one week after the last (the 14th) blood collection. We successfully performed echocardiography-guided closed-chest intraventricular blood sampling, with an average operating time (88±19)s per mouse, and a maximum blood volume (1.43±0.11)ml per mouse. In the repeated 0.5 ml blood collection group, heart rate, left ventricular ejection fraction, left ventricular fractional shortening, left ventricular end-diastolic dimension and left ventricular posterior wall end-diastolic thickness remained uncganged before the first blood collection and after 4 weeks of repeated blood collection (all 0.05). No death in the repeated 0.5 ml blood collection group. However, in the 0.75 ml blood collection group, two mice died before the end point. The echocardiography-guided closed-chest intraventricular blood sampling is a safe, minimally invasive, convenient and efficient method, and can be used repeatedly for long-term blood collection in mice.