Understanding the role of signals involved in wounding responses allows designing and improving preservation technology of fresh-cut products. The present work evaluated the role of ethylene as a signal molecule on phenolic biosynthesis induced by wounding in fresh-cut pitaya fruit at the physiological and gene expression levels. Ethylene pretreatment positively promoted the increases of phenolics and flavonoids of fresh-cut pitaya fruit by improving activities and relative gene expressions of the pivotal enzymes in phenylpropanoid pathway. It accelerated reactive oxygen species (ROS) production at the beginning of storage but caused lower ROS levels during the middle and later periods. Moreover, fresh-cut pitaya fruit pretreated with ethylene showed lower ethylene production rate in comparison with the control, but higher expression levels of ethylene receptor genes (HuETR1 and HuETR2), transcription factor genes (HuEIN3s and HuERF1s) and lower expression levels of the negative ethylene regulator HuCTR1s. On the contrary, the ethylene receptor inhibitor, 1-MCP pretreatment not only reduced the wound-induced phenolic accumulation, prevented the activation of ROS and phenylpropane metabolism, but also inhibited the up-regulation of HuETR1, HuETR2, HuEIN3s and HuERF1s. Taken together, our present work suggested that ethylene pretreatment induced phenolic biosynthesis in fresh-cut pitaya fruit by regulating ethylene signaling pathway.