Choosing 8~10 days cultured hippocampal neurons of SD rat, using Calcium Orange AM and DAF-FM diacetate as the fluorescent indicators of intracellular Ca2+ and nitric oxide(NO), simultaneous detection of intracellular Ca2+ and NO was proposed by double-label method on laser scanning confocal microscope (LSCM). The dyeing process includes two steps and “Two Track” mode of LSCM is applied to realize simultaneous detection of intracellular Ca2+ and NO through quickly switching excitation wavelengths. The experiment results show that there is no cross talk between two dyes and the double-label method can reveal the changes of intracellular Ca2+ and NO concentrations under the stimulation of N-methyl-D-aspartate (NMDA), quite consistent with the results of respective single-label experiments. The analysis of slicing image sequences of double-labeled neurons shows that both Ca2+ and NO are mainly located in the center area of cell bodies, while their distribution details are different. The results suggest that the double-label method can simultaneously detect the intracellular Ca2+ and NO in cultured hippocampal neurons and thus provide an approach to investigate the roles of Ca2+ and NO in neurons as well as the interaction between them.