Two-photon fluorescence microscopy (TPFM) provides key advantages over conventional fluorescence imaging techniques, namely, increased penetration depth, lower tissue autofluorescence and self-absorption, and reduced photodamage and photobleaching and therefore is particularly useful for imaging deep tissues and animals. Enzyme-detecting, small molecule probes provide powerful alternatives over conventional fluorescent protein (FP) based methods in bioimaging, primarily due to their favorable photophysical properties, cell permeability, and chemical tractability. In this article, we report the first fluorogenic, small molecule reporter system (Y2/Y1) capable of imaging endogenous phosphatase activities in both live mammalian cells and Drosophila brains. The one and two photon excited photophysical properties of the system were thoroughly investigated, thus confirming the system was indeed a suitable Turn-ON fluorescence pair for TPFM. To our knowledge, this is the first enzyme reporting two-photon fluorescence bioimaging system which was designed exclusively from a centrosymmetric dye possessing desirable two-photon properties. By conjugation of our reporter system to different cell penetrating peptides (CPPs), we were able to achieve organelle- and tumor cell specific imaging of phosphatase activities with good spatial and temporal resolution. The diffusion problem typically associated with most small molecule imaging probes was effectively abrogated. We further demonstrated this novel two photon system could be used for imaging endogenous phosphatase activities in Drosophila brains with a detection depth of >100 mu m.