The tissue-engineered blood vessel (TEBV) has been developed and used in cardiovascular disease modeling, preclinical drug screening, and for replacement of native diseased arteries. Increasing attention has been paid to biomechanical cues in TEBV and other tissue-engineered organs to better recapitulate the functional properties of the native organs. Currently, computational fluid dynamics models were employed to reveal the hydrodynamics in TEBV-on-a-chip. However, the biomechanical wall stress/strain conditions in the TEBV wall have never been investigated. In this paper, a straight cylindrical TEBV was placed into a polydimethylsiloxane-made microfluidic device to construct the TEBV-on-a-chip. The chip was then perfused with cell culture media flow driven by a peristaltic pump. A three-dimensional fluid–structure interaction (FSI) model was generated to simulate the biomechanical conditions in TEBV and mimic both the dynamic TEBV movement and pulsatile fluid flow. The material stiffness of the TEBV wall was determined by uniaxial tensile testing, while the viscosity of cell culture media was measured using a rheometer. Comparison analysis between the perfusion experiment and FSI model results showed that the average relative error in diameter expansion of TEBV from both approaches was 10.0% in one period. For fluid flow, the average flow velocity over a period was 2.52 cm/s from the FSI model, 10.5% higher than the average velocity of the observed cell clusters (2.28 mm/s) in the experiment. These results demonstrated the facility to apply the FSI modeling approach in TEBV to obtain more comprehensive biomechanical results for investigating mechanical mechanisms of cardiovascular disease development.