Heparin, a naturally occurring glycosaminoglycan, is renowned for its potent anticoagulant properties, which are critical for various medical applications. A significant determinant of its anticoagulant activity is the degree of 3-O-sulfation. Gaining insight into the substrate binding characteristics of 3-O-sulfotransferase-1 (3-OST-1) could enhance our understanding of the sulfotransferase family and facilitate the enzymatic preparation of heparin. This study aimed to identify mutants of 3-OST-1 with improved catalytic activities through a rational design. The enzyme activities of the mutants W72R and H144R were recorded at 26.40 and 17.21 U/L, respectively, representing increases of 1.7 and 1.1 times compared to the wild-type (WT) 3-OST-1. Notably, the enzyme activity of the double mutant W72R/H144R reached 34.41 U/L, which is 2.2 times greater than that of the WT. The heparin modified by the 3-OST-1 mutants exhibited superior anticoagulant properties compared with those modified by the WT, with W72R/H144R demonstrating the highest anticoagulant potency. Furthermore, enzyme kinetic assays and molecular dynamics simulations illustrated that the enhanced catalytic activity of the mutant enzyme resulted from an increased affinity for the substrate.