We report a sensitive and selective method for determination of boron in uranium samples by Spectro fluorimetry in synchronous derivative mode. This method is based on the complexation of non-fluorescent boron with fluorescent chromotropic acid to form fluorescent boron–chromotrope complex. The spectrum of native fluorescence of chromotropic acid seriously over laps with that of the complex and hence, synchronous derivative mode was employed in which physical separation of excess ligand and complex is not necessary. With the optimized experimental and instrumental conditions, limit of detection obtained is 2ngmL^-1. The linear concentration range is 5–100ngmL^-1 with regression coefficient better than 0.997. The precision is better than 5% at 10ngmL^-1 level and 3% at 50 ngmL1 level (n=9).  Fluorescence quenching by residual matrix elements in the final sample solution is corrected by slope-ratio method. The method is validated with reference materials and successfully applied to the uranium nuclear fuels with the accuracy of ±10%.The proposed method reduces sample size requirement; there by reducing load of uranium recovery from analytical waste in case of enriched uranium based samples.