A synthetic strategy of a three-component spiro-pyrrolidine compound based on benzofuran via an [3+2] azomethine ylide cycloaddition reaction is reported herein. Under mild optimal conditions, this reaction can quickly produce potentially bioactive compounds with a wide range of substrates, high yield, and simple operation. The desired products were obtained with a yield of 74–99% and a diastereomeric ratio (dr) of >20:1. Subsequently, the inhibitory effects of the compounds on the cell viability of the human cancer cell line HeLa and mouse cancer cell line CT26 were evaluated. Compounds 4b (IC50 = 15.14 ± 1.33 µM) and 4c (IC50 = 10.26 ± 0.87 µM) showed higher antiproliferative activities against HeLa cells than cisplatin (IC50 = 15.91 ± 1.09 µM); compounds 4e (IC50 = 8.31 ± 0.64 µM) and 4s (IC50 = 5.28 ± 0.72 µM) exhibited better inhibitory activities against CT26 cells than cisplatin (IC50 = 10.27 ± 0.71 µM). The introduction of electron-donating substituents was beneficial to the inhibitory activities against cancer cells. Molecular docking simulations revealed that 4e and 4s may exert corresponding bioactivities by binding to antitumor targets through hydrogen bonds, providing a new approach for discovering spiro-heterocyclic antitumor drugs.