One of the main challenges for successful pharmaceutical application of Catalase (CAT) is maintaining its stability. Physical immobilization of CAT through nano-encapsulation was proposed to resolve this challenge. CAT encapsulating niosomes (e-CAT) were prepared using Brij 30, 52, 76, 92, and 97 in the presence of cholesterol (Ch) by thin film hydration method. Niosomes were characterized for encapsulation efficiency % (EE), size, poly-dispersity index (PI), and morphology. Kinetic parameters, pH optimum, thermal stability, and reusability of CAT were determined. The influence of optimized e-CAT dispersion onto thermally injured rat skin was evaluated. Results revealed that encapsulation enhanced CAT catalytic efficiency (V/K). Free CAT and e-CAT had pH optimum at 7.0. e-CAT exhibited improved thermal stability where it retained 50% residual activity at 60 °C. Free CAT lost its activity after three consecutive operational cycles; however, e-CAT retained 60% of its initial activity following 12 cycles. After 24 h of topical application on thermal injury, a significant difference in lesion size was observed with e-CAT compared with the control group. Based on these encouraging results, CAT immobilization demonstrated a promising novel delivery system that enhances its operational stability. In addition, nano-encapsulated CAT can be anticipated to be beneficial in skin oxidative injury.