This work exhibits the implementation of ultrasound technology in solvent-free synthesis study of n-butyl palmitate using Fermase CALB™10000. Sequential experimental design study was instrumental in determining the most significant process variables. The effect of acid-alcohol molar ratio, enzyme dose, temperature, power and duty cycle on the reaction kinetics was studied. Highest conversion of ~ 96.6% was observed in 50 min at 1:1 molar ratio of palmitic acid to n-butanol, 70 °C temperature, 4% w/w enzyme loading, 40 W power, 70% duty cycle, 25 kHz frequency and 100 rpm speed of impeller. Ping-pong bisubstrate model showed the best fit with kinetic parameters, V = 21.88 M/min/g catalyst, K = 0.011 M, K = 8.74M, K = 0.014M, K = 0.00036M and SSE = 0.0000193. Ultrasound reduced the reaction time by over 70%. The enzyme was reusable for four successive cycles after which it showed decline in conversion.