We report a systematic investigation into the optimization of ZnO nanofiber-based NO gas sensors through precise control of structural parameters. By employing electrospinning technique, we fabricated ZnO nanofibers with controlled diameters (160–310 nm) and thicknesses (19–25 μm), enabling detailed analysis of structure–property relationships in gas sensing performance. The sensors exhibited optimal performance at 200 °C operating temperature, with the thinnest membrane (160 μm) and smallest fiber diameter (9.52 μm) demonstrating superior sensing capabilities. Under these optimized conditions, the sensor achieved a remarkable sensitivity of 25 (Ω/Ω) toward 500 ppb NO gas with a notably fast recovery time of 191 s. Structural characterization revealed that reducing membrane thickness by 30% enhanced sensitivity by 96%, attributed to increased pore area accessibility. In addition, decreasing nanofiber diameter by 90% resulted in a twofold improvement in NO gas sensitivity. The sensing mechanism was elucidated through energy band analysis, revealing the critical role of electron depletion layer modulation at the gas–solid interface. The sensors demonstrated excellent selectivity against common interferents including ethanol, isopropanol, and acetone, with NO response approximately 84 times greater than these compounds. This study provides crucial insights into the rational design of metal oxide nanofiber architectures for enhanced gas sensing performance, offering potential applications in both industrial and biomedical monitoring systems.