Objective To investigate the expression of HSP27 protein and CD34 in retinoblastoma (Rb), and the correlation with its role in angiogenesis, the degree of differentiation and the optic nerve invasion of Rb. Methods Expression of HSP27 and CD34 in Rb tissues of 36 cases was analyzed by using a PV9000 two-step immunohistochemical method. Microvessel density (MVD) was detected by calculating the presence of CD34 positive vessels. Results The expression of HSP27 in a normal retina was located in the ganglion cell layer, and a strong positive expression of HSP27 proteins in Rb cells was mainly located in the endochylema. The positive rates of HSP27 and CD34 in Rb were 69% and 100%, respectively, and expression was significantly related to the degree of differentiation of Rb (P＜0.01 for all). The expression of HSP27 and MVD marked by CD34 was significantly related to the nerve infiltration of Rb (P＜0.01). The expression of HSP27 in Rb was markedly correlated with the expression of MVD (r=0.4659, P＜0.01). Conclusion The determination of HSP27 and MVD marked by CD34 are of significance for evaluating the histologic characteristics and biological behavior of Pb. HSP27 is closely correlated with the invasion of the tumor.