An ultraviolet (UV) irradiation-based in situ silver nanoparticle (AgNP) synthesis approach has drawn significant attention for functionalizing a great variety of biomaterials. Here, we designed an AgNP-functionalized 3D-printed polylactic acid (PLA) composite scaffold with a green physical approach by employing the UV irradiation (1, 2, and 3 h) method without using any reducing agent or heat treatments. In situ AgNP synthesis was performed under different UV exposure times. The zeta sizer analysis results demonstrated that AgNPs were highly monodisperse with the particle size of 20 ± 2.2, 30 ± 3.6, and 50 ± 4.8 nm under various UV light exposure times. In situ synthesis of AgNPs on 3D-printed PLA scaffolds significantly changed the surface hydrophilicity of the 3D-printed scaffolds. These results showed that UV irradiation-based in situ AgNP synthesis on 3D-printed PLA scaffolds can be useful in various biomedical applications, such as cell culture scaffolds, biosensors, and wound healing applications.