Trifoliate orange (Poncirus trifoliata (L.) Raf.) is a conventional citrus rootstock, and its resistance to abiotic stress is critical to citriculture. By using high-throughput RNA sequencing, this study unfolded the whole transcriptome expression profile of trifoliate orange seedlings under osmotic stress (-1.56 MPa) mediated by polyethylene glycol. Based on the morphologic observation and physiological measurements, the seedlings were subjected to remarkable water deficit under 1.5, 6 and 24 h of osmotic stress (OS1.5, OS6 and OS24) but recovered after 4 h of re-watering (RE4). Totally, 9756/11,791, 327/435, 69/95 and 181/102 differentially expressed (DE) protein-coding mRNAs, long non-coding (lnc)RNA, circular (circ)RNA and micro (mi)RNA were identified in leaf/root at 0 h of osmotic stress (OS0), OS1.5, OS6 and OS24 and RE4. With over representation analysis for the DEmRNAs of OS1.5/OS0, OS6/OS0, OS24/OS0 and RE4/OS24, there were 70/95 enriched Gene Ontology terms and 16/28 Kyoto Encyclopedia of Genes and Genomes pathways in leaf/root, thereby displaying diverse biological processes, molecular functions and pathways. With k-means cluster analysis, eight DEmRNA clusters with different expression patterns were classified. In addition, some important molecular players in abscisic acid signaling, mitogen-activated protein kinase cascade, proline biosynthesis and polyamine synthesis were revealed and discussed. Ptrif.0009s2070 was identified as the ortholog of RD29B in trifoliate orange, which is the landmark gene under water deficit and ABA treatment. The targets of DElncRNAs and miRNA and the source genes of DEcircRNAs were identified through bioinformatics approaches. Last, competing endogenous RNA (lncRNA/circRNA/mRNA)-miRNA regulatory network with 503/138 nodes including 30/4 hub miRNAs and 497/108 connections and were constructed in leaf/root. Our results benefit the understanding in osmotic stress response and abiotic stress-resistance gene mining in trifoliate orange.