In order to establish an efficient method for the determination of L-carnitine in infant formula for special medical purposes, the spectrophotometric method for the determination of L-carnitine in foods for infants and young children and dairy products specified in the national standard GB 29989—2013 was optimized with respect to standard curve preparation, sample pretreatment and enzyme preparation. The results showed that under the following conditions: using the optimized standard curve, enzyme extraction at a rotation speed of no less than 6 000 r/min, and hydrolysis of 5 g of homogeneous milk powder (accurate to 0.000 1 g) with 0.25 g of α-amylase at 50 ℃ for 30 min, the optimized method exhibited high precision and spiked recovery. This method could overcome the problems encountered in the determination of L-carnitine in infant formula for special medical purposes by the traditional method such as cloudy filtrate, the dark color of prepared samples that could interfere with the determination of absorbance and low spiked recovery, being suitable for the large-scale determination of L-carnitine in amino acid-based infant formula, extensively hydrolyzed protein infant formula and partially hydrolyzed protein infant formula for special medical purposes.