Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.