An R-3-aminobutyric acid preparation method with high efficiency and high stereoselectivity. The method comprises using aspartase with stereoisomeric catalytic activity derived from Escherichia coli to efficiently convert butenoic acid into R-3-aminobutyric acid. After only 24 h of reaction, the conversion rate is as high as ≥ 98%, and the ee value is ≥ 99.9%. The conversion efficiency is greatly improved, the reaction time is shortened, and the production costs are reduced. The method features a high yield, a high conversion rate, low costs, a short production cycle, a simple process, ease of enlargement, suitability for mass production and the like.