A highly sensitive capillary electrophoretic profiling of nicotine (NIC) and nornicotine (NNIC) was developed and applied to mushrooms with amperometric detection (AD). Effects of the experimental factors including detection potential, separation parameters, and sample pretreatment conditions were investigated. Under the optimal conditions, the electrophoretic analysis of NIC and NNIC was achieved within 8 min on a pencil carbon disc working electrode at 0.95 V, which was lower than those reported previously. Good calibration curves were obtained in 0.01-2.0 g/mL and 0.02-3.0 g/mL with the LOD of 2 ng/mL and 5 ng/mL for NIC and NNIC, respectively. The feasibility of the resultant method was verified. Average recoveries of different fortified levels ranged in 80.7-86.0% and 94.0-98.6% for NNIC and NIC were gained, respectively. Applied to a range of mushrooms (Boletus edulis and Lentinus edodes), the NIC contained naturally was successfully found in the level of 19.71-79.20 g/kg. The results obtained with CE-AD method were acceptable and close to that of HPLC-MS.