Total exposure assessment can be performed through biological monitoring. While blood and urine are commonly used matrices, hair represents an innovative alternative due to its ease of collection and its ability to reflect long term exposure. However, its applicability remains limited by insufficient data and unclear performance. In this study, hair, serum, and urine samples from 62 adults were analyzed for ten organophosphorus flame retardants (OPFRs). Detection frequencies, concentration levels, and relationships between matrices were evaluated using descriptive statistics, correlation, and clustering analyses. The effects of hair washing and urinary dilution corrections were also assessed. A greater number of compounds were detected in hair (7 compounds) compared to urine (5 metabolites only) and serum (2 compounds), indicating higher sensitivity of hair for OPFR detection. Hair concentrations were independent of gender, and strongly correlated between washed and unwashed samples, although washing significantly reduced measured levels for parent OPFRs. Importantly, no significant correlations were observed between hair and urine (DPP, BCEP) or serum (TCPP), suggesting that hair reflects different exposure information rather than duplicating conventional matrices. These findings demonstrate that hair provides complementary and more comprehensive exposure information, supporting its use alongside urine and serum to improve OPFR biomonitoring in epidemiological studies.