The depletion of fossil fuels and their associated environmental concerns necessitate the exploration of sustainable alternatives. 5-Hydroxymethylfurfural (5-HMF), a versatile platform chemical derived from biomass, holds significant potential for the production of biofuels, industrial intermediates, and polymers. This study employs a factorial experimental design to investigate the impact of fructose concentration, organic-to-aqueous phase ratio, and reaction time on 5-HMF yield using a biphasic system with a cation exchange resin catalyst. Optimal conditions predicted by the model, including a 100 g/L fructose solution, an organic-to-aqueous phase ratio of 8.36:1, and a reaction time of 6.91 min, were validated experimentally, resulting in a 73.45% 5-HMF yield. Subsequent purification steps, involving activated carbon adsorption for the organic phase and a two-stage extraction with butanol and NaCl for the aqueous phase, achieved 92.63% and 92.13% purity and recovery, respectively. These findings offer valuable insights for the efficient production of 5-HMF.