Shoot multiplication induced by exogenous cytokinins (CKs) has been commonly used in Phalaenopsis micropropagation for commercial production. Despite this, mechanisms of CKs action on shoot multiplication remain unclear in Phalaenopsis. In this study, we first identified key CKs metabolic genes, including six isopentenyltransferase (PaIPTs), six cytokinin riboside 5′ monophosphate phosphoribohydrolase (PaLOGs), and six cytokinin dehydrogenase (PaCKXs), from the Phalaenopsis genome. Then, we investigated expression profiles of these CKs metabolic genes and endogenous CKs dynamics in shoot proliferation by thidiazuron (TDZ) treatments (an artificial plant growth regulator with strong cytokinin-like activity). Our data showed that these CKs metabolic genes have organ-specific expression patterns. The shoot proliferation in vitro was effectively promoted with increased TDZ concentrations. Following TDZ treatments, the highly expressed CKs metabolic genes in micropropagated shoots were PaIPT1, PaLOG2, and PaCKX4. By 30 days of culture, TDZ treatments significantly induced CK-ribosides levels in micropropagated shoots, such as tZR and iPR (2000-fold and 200-fold, respectively) as compared to the controls, whereas cZR showed only a 10-fold increase. Overexpression of PaIPT1 and PaLOG2 by agroinfiltration assays resulted in increased CK-ribosides levels in tobacco leaves, while overexpression of PaCKX4 resulted in decreased CK-ribosides levels. These findings suggest de novo biosynthesis of CKs induced by TDZ, primarily in elevation of tZR and iPR levels. Our results provide a better understanding of CKs metabolism in Phalaenopsis micropropagation.