In this study, the fatigue properties of carbon fiber-reinforced polymer (CFRP) composite laminates were investigated, specifically focusing on the incorporation of 100-µm polysulfone (PSU) nanofibers as an interleaving material. The PSU nanofibers were produced using the electrospinning technique. Both quasi-static and fatigue tests were conducted on both the reference specimens and the modified specimens to evaluate their mode-I performance. The results revealed an 85% increase in fracture toughness (G_IC) under quasi-static testing. The fatigue plots revealed a noteworthy reduction in the fatigue crack growth rate (da/dN) for the modified specimens due to new toughening mechanisms. Scanning electron microscopy (SEM) demonstrated that, the PSU nanofiber became melted and distributed in the interface, leading to phase separation and a sea-island structure. The presence of PSU microspheres caused crack deflection during delamination, which resulted in increased fracture and fatigue resistance.