We developed a novel method to fabricate a crab-like microrobot that can actuate for a long period in a physiological condition. The microrobot backbone was built with a biocompatible and elastic material—polydimethylsiloxane (PDMS)—by using a specially designed 3D molding aligner, and consisted of three strips of PDMS “legs” connected across a “body.” Cardiomyocytes were then plated on the grooved top surface of the backbone, resulting in a high concentration of pulsating cells. These key techniques enabled the microrobot to walk continuously for over ten days. The performance of our crab-like microrobot was measured at an average velocity of 100 µm s−1, and the estimated total distance it travelled was 50 m over a one-week period. Thus, we have demonstrated for the first time a walking robot that exhibited reliable and long-term actuation performances.