Abstract: Virulence of Nomuraea riley CQNr01 against the 2^nd and 4^th instar larvae of Spodoptera litura was investigated. Spore germination and appressorium formation on isolated epidermis of S. litura was observed by fluorescence microscopy. Results indicated at the concentration of 1×10⁸ and 1×10⁹ spores·mL^-1, the mortality of the 2^nd instar larvae caused by N. riley CQNr01 was 86.58% and 89.91%, respectively, while the mortality of the 4^th instar larvae was only 13.33% and 16.67%. Under fluorescence microscopy, spores of CQNr01 were found adhered to isolated epidermis of the 2^nd and the 4^th instar larvae, and spores germinated within 24 h post inoculation, while adhesion strength of the 2^nd instar larvae was stronger. The germ tubes grew longer and the germination rate became larger as the inoculation time went on. However, under the same inoculation time, the germination rate and the number of appressorium on the 2^nd instar larvae significantly exceeded to those on the 4^th instar larvae. The study showed that virulence of N. riley CQNr01 against the 2^nd and the 4^th instar larvae was significantly different and the epidermis may be the main reason.