Phytosterols are regarded as compounds able to reduce total and low-density lipoprotein cholesterol in the blood, and their esterified derivatives could help to improve the effectiveness of this function. In the present study, the water/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/isooctane reverse micelle (RM) system was set up as a reaction medium for Candida rugosa lipase AY30 (CRL AY30) to synthesize β-sitosterol laurate (β-SLE). The product was identified by TLC, FT-IR, and HPLC-APCI-QqQ-MS/MS and quantified by HPLC. Through stepwise optimization, it was found that CRL AY30 had the highest activity in the water/AOT/isooctane RM system where 50 mM PBS with a pH of 7.5 was adopted as water core to carry CRL AY30, and the proportion of [CRL AY30] (mg/mL), [water] (mM), and [AOT] (mM) was set in 3:375:25, respectively, in isooctane. After screened with single-factor experiments, the esterification reaction conditions in the CRL AY30-water/AOT/isooctane RM system were further optimized by the response surface method as follows: the mole ratio of β-sitosterol to lauric acid of 1:3.5 (25 mM β-sitosterol), the enzyme load of 18% (w/w total reactants), the reaction temperature of 47 °C, and the reaction time of 48 h. As a result, the maximum esterification rate was up to 88.12 ± 0.79%.