The primo vascular system (PVS) has been difficult to detect due to its small diameter and translucent features of the thread-like network. Thus, the developed methods to find and take out PVS were to use contrast-enhancing dyes including Alcian blue, Trypan blue, and Janus green B. To use as a detector to PVS and a biological tool for functional study of PVS, monoclonal antibodies (mAbs) against PVS of rat were generated by various techniques, such as cell fusion, enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), screening of hybridoma, immunofluorescence microscopy (IF). Among 16 mAbs generated, 4 representative mAbs were characterized with their specificities in ELISA, WB, and IF. α-rPVS-m1-1 and α-rPVS-m4-6 had strong binding affinities to PVS in both ELISA and WB but did not show specificities at all in IF. On the contrary, α-rPVS-m3-2 and α-rPVS-m3-4 almost did not respond in WB but had strong binding affinities in ELISA and specificities in IF. Two mAbs stained predominantly at extracellular matrix and cell membrane of PVS of rat in IF. Thus, α-rPVS-m3-2 and α-rPVS-m3-4 can be used as a tool in discriminating PVS from blood vessel (BV) and lymphatic vessel (LV) and other similar tissues of rat in IF.