Serum albumin is an important carrier in the transport of endogenous and exogenous substances across cell membranes. Small molecule drugs could bind to serum albumin to various extents after intravenous injection, which may affect the bioavailability, metabolism, pharmacological and toxicological potency. Organic selenium (Se) compounds exhibit favorable biocompatibility and low toxicity that attracts increased attention from researchers. Herein, a series of selenadiazole derivatives (4a–e) have been synthesized and their cytotoxicity towards various kinds of cells were evaluated. The results suggested that isopropyl benzo[c][1,2,5]selenadiazole-5-carboxylate (4d) exhibited a broad inhibition spectrum on the proliferation of tested cancer cells, with a relatively higher anticancer activity than other derivatives. We found that the differences in their anticancer activities were attributed to the various binding abilities of the Se compounds toward BSA. The bimolecular quenching constant (K_q), apparent quenching constant (K_SV), effective binding constant (K_A) and binding site number (n) were obtained through fluorescence quenching calculations, which indicated that all compounds could efficiently bind to BSA molecules and the fluorescence quenching mechanism was mainly a static quenching procedure. Moreover, serum albumin was found to interact with selenadiazole derivatives (4a–e), thus promoting the cellular uptake and anticancer activity of the Se compounds. Taken together, this study demonstrated that the synthetic selenadiazole derivatives exhibited high anticancer activity and cellular uptake through delivery by human serum.