The cellular compartmentation induced by self-assembly of natural proteins has recently attracted widespread attention due to its structural-functional significance. Among them, as a highly conserved metabolic enzyme and one of the potential targets for cancers and parasitic diseases in drug development, CTP synthase (CTPS) has also been reported to self-assemble into filamentous structures termed cytoophidia. To elucidate the dynamical mechanism of cytoophidium filamentation, we utilize single-molecule fluorescence imaging to observe the real-time self-assembly dynamics of CTPS and the coordinated assembly between CTPS and its interaction partner, Δ1-pyrroline-5-carboxylate synthase (P5CS). Significant differences exist in the direction of growth and extension when the two proteins self-assemble. The oligomer state distribution analysis of the CTPS minimum structural subunit under different conditions and the stoichiometry statistics of binding CTPS and P5CS by single-molecule fluorescence photobleach counting further confirm that the CTPS cytoophidia are mainly stacked with tetramers. CTPS can act as the nucleation core to induce the subsequent growth of the P5CS filaments. Our work not only provide evidence from the molecular level for the self-assembly and coordinated assembly (coassembly) of CTPS with its interaction partner P5CS in vitro but also offer new experimental perspectives for the dynamics research of coordinated regulation between other protein polymers.