Cordyceps javanica, a broad-spectrum insecticidal fungus, is extensively utilized to counteract agricultural and forest pests. Effectors are crucial in the interactions between entomopathogenic fungi and their host insects. Candidate effectors were selected from 11142 predicted proteins in the whole published genome of C. javanica to search for their candidate effectors for functional analysis. It was predicted with bioinformatics software, including SignalP v5.0, TMHMM v2.0, TargetP v2.0, and Protcomp v9.0. A total of 611 proteins which might localize in the cytoplasm have been selected. Among them, subsequent screening unveiled 348 sequences with a high cysteine content and 117 proteins featuring repeated sequences. A total of 57 protein encoded were compared with the pathogen-host interaction (PHI) database and identified, from which 10 candidate effectors were identified. Finally, four cytoplasmic effector factors related to pathogenicity were screened out based on mutant phenotypes prediction. The expression patterns of encoding genes of two effectors (the cell wall protein TQW04886 and the hydrophobic protein TQW02068) during the transition from vegetative growth to reproductive growth were investigated by quantitative real-time PCR. The results showed that these two genes were highly expressed in C. javanica during the sporulation stage. This study lays the foundation for a better understanding of molecular mechanism in the interaction between the pathogen C. javanica and its host, and provides reference for the prediction of effectors in other entomopathogenic fungi.