Gastric cancer (GC), arising from gastric mucosal cells, necessitates innovative treatment strategies beyond conventional surgical approaches. While 5-fluorouracil (5-FU) has demonstrated efficacy in various cancers, its lack of selectivity for cancer cells and limited half-life pose challenges. This study focuses on assessing the therapeutic potential of 5-FU-loaded L-polylactic acid (PLLA) nanofibers (NFs) for targeted GC treatment. The preparation of 5-FU/PLLA NFs involved refining the drug delivery approach to enhance drug impact on GC cell proliferation and apoptosis. Utilizing an organic phase separation methodology, 5-FU was incorporated into PLLA NFs, and the NF morphology was examined using scanning electron microscopy, with optical microscopy used for diameter measurement. Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) explored binding state and PLLA crystallinity. Drug loading (DL) capacity and in vitro release characteristics were evaluated by UV-visible spectrophotometry, while NF degradation and stability were assessed. The human gastric adenocarcinoma (AGS) cell line was employed in cell experiments, with three groups: normal culture (Normal group), single drug treatment with 5-FU (5 μmol/L, 5-FU group), and 5-FU-loaded PLLA group (5-FU/PLLA group) containing 5 μmol/L 5-FU. Cell Counting Kit-8 gauged cell proliferation and viability, and Annexin V-FITC/PI assay determined cell apoptosis. Results revealed a (1,230.8±18.9) nm diameter for 5-FU/PLLA NFs with 18.3% crystallinity. FTIR and DSC analyses indicated a simple physical mixture of 5-FU and PLLA in 5-FU/PLLA. DL capacity was (18.1±2.3)%, with a DL efficiency of (92.5±9.4)%. in vitro release performance of 5-FU/PLLA surpassed that of raw 5-FU. The mass loss rate of 5-FU/PLLA was consistent across different pH buffer solutions, with a stable drug release (DR) rate over various storage times. In cell experiments, both 5-FU and 5-FU/PLLA groups exhibited reduced proliferation and viability compared to the Normal group, with higher apoptosis rates (P < 0.05). Furthermore, the 5-FU/PLLA group showed decreased proliferation and viability compared to the 5-FU group, accompanied by higher apoptosis rates (P < 0.05). In conclusion, 5-FU-loaded PLLA NFs, with excellent DR properties, demonstrated significant inhibitory and cytotoxic effects on GC cells.