Three fluorenone-derived two-photon fluorescent probes (TK) targeting the lysosomes (TK-Lyso) and mitochondria (TK-Mito1 and TK-Mito2) were synthesized by introducing different diphenylamine moieties on fluorenone-core. The TK dyes showed high biocompatibility and long-term retention, low cytotoxicity, large Stoke’s shift and good fluorescence quantum yield. The results of the present work disclose a class of organic dyes with potential wide applications as specific and efficient lysosome and mitochondria probes in the study of various biological processes.