Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study， we focused on AoOSBP1， one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1， in both gene and protein levels， was stably expressed throughout the developmental stages， while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore， indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1， namely OSBP-related domain (ORD)， was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol， and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast， MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols， plays an underlying role in sterols transportation， and may participate in spore formation.