The association of 17β-hydroxysteroid dehydrogenase 10 (HSD10) with β-amyloid in the brain is known to contribute to the progression of Alzheimer’s disease. Further, it has been shown that the interaction between the purified HSD10 and β-amyloid inhibits its enzymatic activity. However, to date no system has been developed to enable the study of HSD10 activity in intact living cells. To address this significant shortcoming, we have developed a novel fluorogenic probe, (−)-cyclohexenyl amino naphthalene alcohol [(−)-CHANA], to observe and measure the activity of HSD10 in living cells. The oxidation of (−)-CHANA by HSD10 results in the production and accumulation of a fluorescent product, which can be measured using real-time fluorescence microscopy. This compound permits the measurement of mitochondrial HSD10 activity and its inhibition by both a small molecule HSD10 inhibitor and by β-amyloid, in living cells. Herein, we define the parameters under which this probe can be used. This compound is likely to prove useful in future investigations aimed at developing therapeutic compounds targeting the HSD10-β-amyloid association.