The pursuit of sustainable materials for additive manufacturing has drawn significant attention from both industry and academia. This study focuses on the development and optimization of jute–polylactic acid (PLA) composite filaments as a biodegradable alternative to petroleum-based thermoplastics for 3D printing. Key challenges, including filament thickness, uniformity, brittleness, and printability, were addressed by optimizing the material composition. The Taguchi design of experiment was employed with three factors and three levels. Filaments were produced using PLA blended with 5 wt%, 7.5 wt%, and 10 wt% jute fiber via a single-screw extruder under three different outlet temperatures (150℃, 155℃, and 160℃). An automated winding system, incorporating microprocessor control, tension regulation, and a diameter measuring sensor, was designed to enhance filament uniformity. The printability of the jute filament is checked based on the product surface morphology by changing the nozzle size of the 3D printer. After optimizing the nozzle size, the filaments were mechanically and thermally characterized. Mechanical characterization, including tensile (ASTM D638) and flexural (ASTM D790) strength tests, demonstrated improved strength properties with increasing jute reinforcement percentage, particularly at 10 wt.%. However, at 10 wt.% jute, increased brittleness and extrusion discontinuity were observed. At higher temperatures, this discontinuity was minimized. Thermogravimetric Analysis (ASTM D3850), water uptake (ASTM 570), and microscopic imaging further supported these results, revealing enhanced thermal stability but increased moisture absorption with higher jute content in the filament.