One-dimensional γ-Al2O3 nanofibers were modified with Nb2O5 to be used as an efficient heterogeneous catalyst to catalyze biomass into 5-hydroxymethylfurfural (5-HMF). At low Nb2O5 loading, the niobia species were well dispersed on γ-Al2O3 nanofiber through Nb–O–Al bridge bonds. The interaction between Nb2O5 precursor and γ-Al2O3 nanofiber results in the niobia species with strong Lewis acid sites and intensive Brønsted acid sites, which made 5-HMF yield from glucose to reach the maximum 55.9~59.0% over Nb2O5-γ-Al2O3 nanofiber with a loading of 0.5~1 wt% Nb2O5 at 150 °C for 4 h in dimethyl sulfoxide. However, increasing Nb2O5 loading could lead to the formation of two-dimensional polymerized niobia species, three-dimensional polymerized niobia species and crystallization, which significantly influenced the distribution and quantity of the Lewis acid sites and Brönst acid sites over Nb2O5-γ-Al2O3 nanofiber. Lewis acid site Nbδ+ played a key role on the isomerization of glucose to fructose, while Brønsted acid sites are more active for the dehydration of generated fructose to 5-HMF. In addition, the heterogeneous Nb2O5-γ-Al2O3 nanofiber catalyst with suitable ratio of Lewis acid to Brönsted sites should display an more excellent catalytic performance in the conversion of glucose to 5-HMF.