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Abstract Nowadays, monitoring of volatile organic compounds (VOCs) is very important in various domains. In this work, we aimed to develop sensitive olfactory biosensors using odorant binding proteins (OBPs) as sensing materials. Three rat OBP3 derivatives with customized binding properties were designed and immobilized on the same chip for the detection of VOCs in solution by surface plasmon resonance imaging (SPRi). We demonstrated that the proteins kept their binding properties after the immobilization under optimized conditions. The obtained olfactory biosensors exhibited very low limits of detection in both concentration (200 pM of β-ionone) and in molecular weight of VOCs (100 g/mol for hexanal). Such a performance obtained with SPRi in solution is especially remarkable. We hypothesized that the binding of VOCs to the active sites of OBPs induced a local conformational change in the proteins. This change would give rise to a variation of refractive index, to which SPRi is extremely sensitive. In addition, the olfactory biosensors showed a high selectivity especially at relatively low VOC concentrations. With optimized regeneration procedures, they also showed very good repeatability not only from measurement to measurement but also from chip to chip with a lifespan up to almost two months. These olfactory biosensors are particularly interesting for trace detection of VOCs in solution. Highlights • Custom-made odorant binding proteins (OBPs) are used for olfactory biosensor development. • SPRi is efficient for the detection of volatile organic compounds in solution. • A local conformational change in OBPs has most likely amplified the optical signal. • The olfactory biosensors are highly sensitive and selective at low concentrations. [ABSTRACT FROM AUTHOR]