Polylactic acid (PLA) is a thermoplastic and biodegradable polyester, largely derived from renewable resources such as corn starch, cassava starch and sugarcane. However, PLA is only soluble in a narrow range of solvents such as tetrahydrofuran, dioxane, chlorinated solvents and heated benzene. The limited choices of solvent for PLA dissolution have imposed singificant challenges in the development of specifically engineered PLA nanofibers with electrospinning techniques. Generally, the electrospun polymeric materials have been rendered with unique properties such as high porosity and complex geometry while maintaining its biodegradability and biocompatibility for emerging biomedical applications. In this study, a new anticancer drug delivery system composed of PLA nanofibers with encapsulated paclitaxel was developed by the electrospinning of the respective nanofibers on top of a spin-coated thin film with the same chemical compositions. Our unique approach is meant for promoting strong bonding between PLA-based nanofibers and their respective films in order to improve the prolonged release properties and composite film stability within a fluctuative phyiochemical environment during cell culture. PLA/paclitaxel nanofiber supported on respective polymeric films were probed by scanning electronic microscope, Fourier transform infrared spectrometer and water contact measurement for determining their surface morphologies, fibers’ diameters, molecular vibrational modes, and wettability, respectively. Moreover, PLA/paclitaxel nanofibers supported on respective spin-coated films at different loadings of paclitaxel were evaluated for their abilities in killing human colorectal carcinoma cells (HCT-116). More importantly, MTT assays showed that regardless of the concentrations of paclitaxel, the growth of HCT-116 was effectively inhibited by the prolonged release of paclitaxel from PLA/paclitaxel nanofibers. An effective prolonged delivery system of paclitaxel based on PLA nanofiber-based film has demonstrated exciting potentials for emerging applications as implantable drug delivery patch in post-surgical cancer eradication.