Intraoperative imaging of living tissue at the cell level by endomicroscopy might help surgeons optimize surgical procedures and provide individualized treatments. However, the resolution of the microscopic image is limited by the motion of living tissue caused by heartbeat and respiration. An active motion compensation (AMC) strategy has been recognized as an effective way to reduce, or even eliminate, the influence of tissue movement for intravital fluorescence microscopy (IVM). To realize the AMC system, a high-speed sensor for measuring the motion of tissues is needed. At present, state-of-the-art commercialized displacement sensors are not suitable to apply in minimally invasive imaging instruments to measure the motion of living tissues because of the size problem, range of measurement or the update rate. In this study, a compact high-speed image-based method for measuring the longitudinal motion of living tissues is proposed. The complexity of the proposed method is the same as that of the traditional wide-field fluorescent microscopy (WFFM) system, which makes it easy to be miniaturized and integrated into a minimally invasive imaging instrument. Experimental results reveal that the maximum indication error, range of measurement and the sensitivity of the laboratory-built experimental prototype is 150 μm, 6 mm and −211.46 mm−1 respectively. Experimental results indicate that the proposed optical method is expected to be used in minimally invasive imaging instruments to build an AMC system.