Nowadays, breast milk is considered as the ideal food for infants owing to the most common oleic acid-palmitic acid-oleic acid (OA-PA-OA) fatty acid distribution of the human milk fat (HMF). This study reports the synthesis of 1,3-dioleoyl-2-palmotoylglycerol (OPO)-rich human milk fat substitutes in a two-step enzymatic acidolysis reaction with Rhizomucor miehei lipase (RML) immobilized on magnetic multi-walled carbon nanotubes(mMWCNTs). The immobilized RML (RML-mMWCNTs) showed better thermal and pH stability, convenient recovery and reusability than the free soluble form. Under optimized reaction conditions (1:8 tripalmitin (PPP)/OA, 10%wt. enzyme, 50 °C, 5 h), PA content at the sn-2 position and OA incorporation at the sn-1,3 positions reached 93.46% and 59.54%, respectively. Comparison tests have also showed that RML-mMWCNTs has better catalytic activity and reusability than the commercial lipase Lipozyme RM IM. The results suggest that RML-mMWCNTs is a promising biocatalyst for the synthesis of OPO-rich TAGs with potential use in infant formulas.