Serotonin (5-hydroxytryptamine) is an indoleamine with important physiological functions in plants, including developmental processes and stress responses. In this study, a simple and rapid bioassay was developed to investigate the inhibition of the fruit browning enzyme, polyphenol oxidase (PPO) via serotonin, employing cut apple discs as well as apple tissue homogenate treated with serotonin. The present study demonstrated that serotonin is significantly effective in inhibiting browning compared to ascorbic acid. Serotonin reduced fruit browning in tissues as well as suppressed the activity of PPO enzyme in the browning model solutions containing epicatechin (Epi) and caffeoyl tartrate (CT) as polyphenol substrates. The determination of enzyme kinetic parameters also suggested that serotonin may act as a putative, non-competitive inhibitor of PPO. Apart from its anti-browning effect, the application of serotonin significantly increased total phenolics content and antioxidant capacity when applied to the apple homogenate. Furthermore, the analysis of MdPPOs showed that five (MdPPO2, MdPPO4, MdPPO6, MdPPO8 and MdPPO10) out of the nine MdPPOs expressed in apple fruits were down-regulated in apple tissues treated with serotonin. To the best of our knowledge, this is the first report documenting a role for serotonin in the biochemical and transcriptional regulation of PPO enzymes and a simple system to study the biochemical control of browning in pome fruits.