A rapid, simple, and inexpensive spectrofluorimetric sensor has been developed for the simultaneous determination of methotrexate (MTX) and folic acid (FA) based on their interactions with hollow carbon dots (HCDs). Since the use of folic acid to cope with the toxic side effects of MTX in patients is essential, the simultaneous determination of these two compounds has been interesting. The results showed that MTX could quench the fluorescence of HCDs with a dynamic quenching mechanism. The sensor exhibited alinear concentration range of 1.0 × 10-6-1.9 × 10-4mol L-1for MTX and 1.5 × 10-5-9.4 × 10-4mol L-1for FA and the obtaineddetection limits for MTX and FA were 1.6 × 10-7and 5.0 × 10-7mol L-1, respectively. The applicability of the method was investigated in the analysis of the urine samples and the partial least squares(PLS) method was used forthe simultaneous determination of MTX and FA.