SSI and GSI in self-incompatibility (SI) plant are mainly controlled by S locus. Camellia oleifera belongs to Late-acting self-incompatibility (LSI) plant, and its self-incompatibility has become a limiting factor to the development of Camellia oleifera industry. To investigate the molecular mechanism of LSI in Camellia oleifera, we designed primers for five S-RNase (CoS-RNase) genes sequences derived from the preliminary study of the annotated genome-wide data of elite variety called‘Huashuo’ and the pistil transcriptomic data of camellia oleifera. By PCR amplification of CoS-RNases f exons rom 10 camellia oleifera varieties, in conjugation with the full-length cDNA of pistil -CoS-RNase, 28 CoS-RNase alleles were annotated. The CoS-RNase gene contains 4 exons and 3 introns, with the cDNA complete sequence of 1141 bp which contains an open reading frame of 717 bp encoding for 238 amino acids. Phylogenetic analysis revealled the highest similarity (98.6%-98.8%) between CoS-RNase with CsS-RNase of tea plant. The CoS-RNases possessed two conserved domains and active sites of T2 RNase protein family, and had the same action sites as in S-RNase of apple and pear: a histidine (His, (His, 119 bit)) site that degraded RNA and a proline (Pro, 156’) site that depolymerized actin. In vitro recombinant protein studies indicated that the recombinant protein CoS-RNase had RNase activity and inhibited pollen tube growth. qRT-PCR analysis showed CoS-RNase was rarely expressed in the pollen while expressed in all other tissues. Morever, the expression pattern of CoS-RNase in styles and in ovaries respectively after self-pollination and outcrossing were consistent with pollen tube growth law. These results implied CoS-RNase as an important factor involved in the SI reaction in Camellia moleifera. Collectively, this study provided a theoretical basis for future deciphering the molecular regulation of self-incompatibility in Camellia oleifera.