AbstractThis work aims at evaluating the potential of Carica papaya lipase (CPL) self‐immobilized in papaya latex as a biocatalyst for the synthesis of human milk fat substitutes (HMFS), to be used as a low‐cost alternative to commercial lipases. Two different CPL preparations, one extracted from the papaya fruit (CPL I) and the other from petiole leaves (CPL II) of papaya tree, were tested as catalysts for the acidolysis between tripalmitin and (i) oleic acid or (ii) omega‐3 PUFA, batchwise, at 60°C, in solvent‐free media. After 24 h, molar incorporation was higher for oleic acid (22.1 mol%) when CPL I was used. This biocatalyst was selected for further studies. RSM was used to model reaction conditions: medium formulation (molar ratio oleic acid/tripalmitin, MR, 1.2:1–6.8:1) and temperature (58–72°C). Acyl migration decreased with MR increase. In batch operational stability assays at 60°C, using MR of 2:1 and 6:1, the highest stability was observed for a MR of 2:1.Practical applications: The use of this biocatalyst is a feasible way to valorize papaya agro‐residues which represent an important environmental problem in the producing countries. The obtained results were rather promising since, with this almost zero‐cost biocatalyst, it was possible to produce a high added‐value product (HMFS). Under optimized conditions, the obtained results were comparable with those obtained with expensive immobilized commercial lipases.