Provided is a method of isolating and purifying biomolecules using a hydrogel, the method including: bring a sample containing charged biomolecules into contact with a hydrogel to bind the biomolecules to the hydrogel; washing the hydrogel bound with the biomolecules; and eluting the bound biomolecules using an elution solvent. According to the method, the use of a hydrogel with a large surface area reduces the isolation time of biomolecules to 5 min or less, an external device such as an electromagnet is not required, and small-sized systems or LOC can be easily implemented due to applicability to microsystems through a polymer patterning technique.