Drosophila fatty acid binding protein isoform B (dFABP) is a member of fatty acid-binding protein family which is discovered in brain tissue. Besides dFABP has approximate 50% sequence similarity to mammalian brain-type FABP (FABP7), it has been reported to be involved in enhancement of long-term memory (LTM) consolidation in Drosophila melanogaster. However, the preference of lipid binding or the possibly molecular mechanism of LTM consolidation mediated by dFABP remains unclear. In this study, we attempt to characterize structural properties and biological functions of dFABP via circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR). Unexpectedly, the results of CD spectra implied that the stability of structure is sensitive to pH value. In accordance with ANS competitive experiment, dFABP has a dominant binding preference toward unsaturated than saturated fatty acids. Furthermore, structural analysis of dFABP/DHA and dFABP/OA complexe models and site-directed mutagenesis experiments indicated that Tyr126 and Arg124 play important roles in ligand binding. Based on the homology sequence alignments and the surface electric analysis of model, we categorize dFABP as group iv which folds fatty acid in U-shaped or highly bent, and transfers lipid in a collision-mediated process. In conclusion, we gain insights into the fatty acid binding affinities and the structural properties of dFABP that pH adjustment and specific residues would cooperatively contribute to understanding long-term memory consolidation.