The solid-liquid ratio and ethanol volume fraction for extraction of total alkaloids from Chelidonium were optimized by uniform design method with total alkaloid content as evaluation index. The purification process was further established via macroporous adsorption resin method by adjusting the diameter-to-height ratio, sample concentration, and eluent volume. The optimal extraction process parameters of total alkaloids from Chelidonium were as follows: solid-liquid ratio of 1:30, 95% ethanol, 3 cycles (1.7 h each). The optimal purification process was as follows: using X-5 macroporous adsorption resin, mass concentration of sample liquid of 0.20 g/mL, diameter-to-height ratio of 1:11, sample liquid pH 7, sample flow rate of 1 BV/h, impurity removal with 6 BV of water, and elution with 10 BV of 95% ethanol at 2 BV/h. The final purity and yield of total alkaloids were 95.10%, and 2.75%, respectively. Pearson correlation analysis revealed a significant positive correlation between alkaloid content and colon cancer cell inhibition rate. These results demonstrate a clear dose-effect relationship between alkaloid content and anti-tumor activity, indicating the feasibility of the extraction and purification process for developing anti-colon cancer drugs.