A novel 1,2,4-triazole-5-thiol derivative was designed through a series of reactions. Firstly, Azo derivative 1 was synthesized by a general method with two steps, a diazotization reaction in an acidic medium, and a coupling reaction in basic conditions. Secondly, Azo-carboxylic acid 1 reacted with SOCl2 to produce acid chloride derivative 2. Then carbothiamide derivative 3 was synthesized through substitution reaction by reacting 2 with 4-phenyl thiosemicarbazide in pyridine. In the next step, carbothiamide 3 was reacted with sodium hydroxide solution to afford target triazole derivative 4. The structures of all synthesized derivatives were confirmed by techniques (1H, 13C NMR, IR). Moreover, the triazole derivative 4 was evaluated for its biological activity as an anticancer, antioxidant, and antibacterial. The results presented that compound 4 has moderate antibacterial activity and a high free radical scavenging effect (IC50 30.2 μg/mL) compared to vitamin C (IC50 21 μg/mL). The anticancer activity results presented that compound 4 has a toxic effect on a colon cancer cell line CaCo-2. The potential effect of compound 4 on the 5lqf protein was examined in silico studies.