Sugarcane molasses is a wealthy source of health beneficial phenolic compounds and exhibited significant biological properties. Present study was designed to assess the inhibitory effect of sugarcane molasses on 4-glucosidase and 4-amylase. Hypoglycemic fractions were extracted using macro-porous and ion exchange resins. The total phenolics content was 0.179 ± 0.003 mg GAE per milligram of extract. Four phenolic acids which include caffeic acid (11.64 mg/g), ferulic acid (10.49 mg/g), chlorogenic acid (1.77 mg/g), and gallic acid (0.87 mg/g) were identified and quantified by high-performance liquid chromatography. The inhibitory activities of sugarcane molasses were 4.693 mg/mL (Km = 1.099 mL/mg) and 4.254 mg/mL (Km 0.238 mL/mg) for 4-glucosidase and 4-amylase, respectively, which revealed that sugarcane molasses could be a useful addition in medicinal preparations as nutraceutical and functional food for diabetic patients.