Abstract: Objective To improve Luo-Ye pump-based stress-forming system and optimize the stimulating effect on smooth muscle cells during cultivation of tissue-engineered blood vessels (TEBV). Methods A new Luo-Ye pump-based TEBV 3D culture system was developed by adding an air pump to the output of the bioreactor. A pressure guide wire was used to measure the stress at different points of the silicone tube inside the TEBV bio-reactor, and fitting curves of the stress changes over time was created using Origin 8.0 software. The TEBVs were constructed by seeding vascular smooth muscle cells (VSMCs) isolated from human umbilical artery on polyglycolic acid (PGA) and cultured under dynamic conditions with 40 mmHg resistance (improved group), dynamic conditions without resistance (control group) or static condition (static group) for 4 weeks. The harvested TEBVs were then examined with HE staining, masson staining, α-SMA immunohistochemical staining, and scanning and transmission electron microscopy with semi-quantitative analysis of collagen content and α-SMA expression. Results The measured stress values and the fitting curves showed that the stress stimuli from the Luo-Ye pump were enhanced by adding an air pump to the output of the bioreactor. Histological analysis revealed improved VSMC density, collagen content and α-SMA expression in the TEBVs constructed with the improved method as compared with those in the control and static groups. Conclusion Adding an air pump to the Luo-Ye pump significantly enhances the stress stimulation in the TEBV 3-D culture system to promote the secretion function of VSMCs.