In this study, the microbial degradation of six pyrethroids (bifenthrin, fenpropathrin, permethrin, alpha-cypermethrin, fenvalerate and deltamethrin) was investigated in soil to develop a new approach to estimate bioavailability of pyrethoids by compound-specific stable isotope analysis (CSIA). After 56-day incubation, the residual concentrations of pyrethroids in unsterilized soil were much lower than those in sterilized soil, indicating that microbial degradation was dominant for the elimination of pyrethroids. Meanwhile, the stable carbon isotope ratios of the six pyrethroids were determined during the degradation. Significant stable carbon isotope fractionation was observed during the microbial degradation of fenpropathrin, alpha-cypermethrin and deltamethrinhe. The enrichment factor epsilon was determined as -1.88 parts per thousand, -1.82 parts per thousand and -2.00 parts per thousand, respectively. A new approach was then developed to estimate the bioavailability of the three pyrethroids by determining stable carbon isotope ratios. The approach established in the study provides an alternative method to dynamically and quantitatively estimate bioavailability of compounds in soil based on CSIA.