The enzymatic transesterification of Atlantic salmon (Salmo salar) oil was carried out using Novozym 435 (immobilized lipase from Candida antartica) to produce biodiesel. A response surface modelling design was performed to investigate the relationship between biodiesel yield and several critical factors, including enzyme concentration (5, 10, or 15%), temperature (40, 45, or 50 °C), oil/alcohol molar ratio (1:3, 1:4, or 1:5) and time (8, 16, or 24 h). The results indicated that the effects of all the factors were statistically significant at p-values of 0.000 for biodiesel production. The optimum parameters for biodiesel production were determined as 10% enzyme concentration, 45 °C, 16 h, and 1:4 oil/alcohol molar ratio, leading to a biodiesel yield of 87.23%. The step-wise addition of methanol during the enzymatic transesterification further increased the biodiesel yield to 94.5%. This is the first study that focused on Atlantic salmon oil-derived biodiesel production, which creates a paradigm for valorization of Atlantic salmon by-products that would also reduce the consumption and demand of plant oils derived from crops and vegetables.