In this work, the spectral responses of hypocrellin B (HB) to the microenvironments of various biomolecules were studied, with human serum albumin (HSA), bovine serum albumin (BSA) and ovalbumin (OVA) used as the models for proteins, sodium alginate (SOA) and hyaluronan (HYA) for polysaccharides and liposomes for lipid membranes. Generally, compared to those in aqueous solution, the absorbance and fluorescence of HB were all strengthened in the model systems except for the fluorescence in HYA. Specially, according to the spectral responses of HB, the microenvironments in biomolecules and liposomes could be set in a sequence of hydrophobic grades, i.e., liposomes > proteins > polysaccharides. Further, R(F/A), a parameter defined as the ratio of the fluorescence intensity to the absorbance, was proposed to identify the microenvironment quantitatively. It was found that the R(F/A) could not only distinguish various types of biomolecules but also identify specific binding from nonspecific binding to proteins or polysaccharides.