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Tissue architecture plays critical roles in the physiological functions of blood vessels. Surface-patterned films are promising to replicate cellular alignment as in the native vessels. However, for vascular tissue engineering (TE) applications, the current surface-patterned films lack structural support for the myoendothelial communications between tunica media and intima. Herein, we report the development of direct microperforation using a femtosecond laser on surface-patterned films for the native-like architecture reconstruction of blood vessels. Poly(ε-caprolactone) (PCL) thin films were surface-patterned with anisotropic microridges/grooves. Direct femtosecond laser ablation further resulted in microscale through-holes for the PCL films, without invasive thermal damage to the ridges/grooves on the nonprocessed surface. Laser fluence and pulse number were observed to significantly influence the microperforation on both hole quality and dimension. The PCL films after direct femtosecond laser microperforation exhibited improved flexible properties, without sacrificing the yield stress. Meanwhile, direct femtosecond laser microperforation resulted in PCL films with hydrophilic permeability to transport nutritional/signaling biomolecules and allowed for heterocellular protrusion ingrowth into the through-holes for physical myoendothelial contacts. Small-diameter vascular TE scaffolds based on the as-fabricated PCL films could enable a hybrid vascular wall construction with aligned stromal multilayers and a confluent endothelium similar to those of the native vascular tissue. These results showed that direct femtosecond laser microperforation could be a reliable approach for producing biomimetic films with through-holes. The developed vascular TE scaffolds with microridges/grooves and through-holes have the potential to offer structural support for vascular architecture reconstruction with the native-like stromal and endothelial components.