Corn starch played an important role in the food industries, while traditional extraction method involved using sulfur dioxide that could easily cause environmental pollution and equipment corrosion. Based on these drawbacks, the main objective of this study was to develop a feasible two-step enzymatic extraction method that could isolate starch from corn kernels and evaluate the properties of corn starch. The physicochemical components of steep liquor during enzymatic extraction were researched, the single-factor experiments and response surface methodology were used to optimize the steeping conditions. As a result, optimized steeping parameters of which the first step time was 30 h, the lactic acid solution concentration was 0.45%, the protease content was 480 U/g, the l-cysteine content was 0.45 g, and the second step time was 4 h. The corn starch yield (65.18%) with enzymatic extraction was higher than that of traditional extraction (54.73%). The solubility and swelling power of all starch samples were raised with the temperature increasing. The starch extracted using the enzymatic method had a higher amylose content (35.18%) and mean diameter (30.99 μm). Compared with commercial corn starch, the starch samples presented nearly identical FT-IR spectra, the same A-type crystalline pattern and morphology, furthermore, a higher resistant starch content was detected in corn starch obtained through extraction methods. This finding will provide useful information to food industries for extraction of corn starch.