With the dwindling availability of fossil fuel resources and the dramatic increase in demand, 5-methoxymethylfurfural (MMF) obtained from renewable biomass resources has great potential as a fossil fuel alternative. In this work, a high yield (80.5%) of MMF was obtained from fructose catalyzed by Amberlyst-15 with the assistance of dimethyl sulfoxide (DMSO) in methanol at 100 degrees C for 10 h. Based on a detailed investigation of the reaction mechanism through the effects of DMSO, Amberlyst-15, reaction time, and reaction temperature on MMF yield and the existence of intermediates proved by FTIR, a two-step pathway was proposed: (1) DMSO and H ion on Amberlyst-15 act as a joint-catalyst [DMSOH](+), facilitating the dehydration of fructose into 5-hydroxymethylfurfural (HMF), and (2) [DMSOH](+) assists the etherification of HMF with methanol into MMF, which is the rate-determining step. This finding expands the concept of biomass conversion into biomass fuels and plays a vital role in catalyst screen and design, reaction medium selection, and industrial applications.