A series of biotinylated camptothecin derivatives were designed and synthesized. The key to the synthesis was achieved by employing an esterification reaction and click chemistry. All of the new derivatives were tested for cytotoxicity against five human tumor cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW480 with IC50 values ranging from 0.13 to 21.53 μM. Most of the derivatives exhibited potent cytotoxicity, especially compound 17 (IC50 = 0.13-3.31 μM) and compound 18 (IC50 = 0.23-1.48 μM), which exhibited the highest potencies. The structure-activity relationships (SARs) of the biotinylated camptothecin derivatives were discussed for exploring novel anticancer agents.