Tumor vaccine therapy offers significant advantages over conventional treatments, including reduced toxic side effects. However, it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods. Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors, thereby limiting their clinical efficacy. In this study, we developed a nanoparticle-based tumor vaccine, OVA@HA-PEI, utilizing ovalbumin (OVA) as the presenting antigen and hyaluronic acid (HA) and polyethyleneimine (PEI) as adjuvants and carriers. This formulation significantly enhanced the proliferation of immune cells and cytokines, such as CD3, CD8, interferon-γ, and tumor necrosis factor-α, in vivo, effectively activating an immune response against B16–F10 tumors. In vivo fluorescence flow cytometry (IVFC) has already become an effective method for monitoring circulating tumor cells (CTCs) due to its direct, noninvasive, and long-term detection capabilities. Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1 (PD-1) antibody. The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone. Additionally, this combination therapy substantially reduced the number of CTCs in vivo, increased the clearance rate of CTCs by the immune system, and slowed tumor progression. These findings greatly enhance the clinical application prospects of IVFC and tumor vaccines.