Combinatorial immuno-cancer therapy has been recognized as a promising approach for efficiently treating malignant tumors. Yet, the development of multifunctional nanomedicine capable of precise tumor targeting, remote activation, and immune-regulating drug delivery remains a significant challenge. In this study, we developed nanoparticles loaded with an immune checkpoint inhibitor (JQ-1) using polypyrrole/hyaluronic acid (PPyHA/JQ-1). These nanoparticles offer active tumor targeting, photothermal tumor ablation using near-infrared light, and laser-controlled JQ-1 release for efficient breast cancer treatment. We varied the molecular weights of HA (6.8 kDa to 3 MDa) in the PPyHA nanoparticles and found that the nanoparticles synthesized using 1 MDa HA, referred to as PPyHA (1 M), showed the most suitable properties, including small hydrodynamic size, high surface HA contents, and colloidal stability. Upon 808-nm laser irradiation, PPyHA/JQ-1 elevated the temperature above 55 °C, which was sufficient for thermal ablation and active release of JQ-1 in the tumor microenvironment (TME). Notably, the controlled release of JQ-1 substantially inhibited the expression of cancer-promoting c-MYC/BRD4 genes. Furthermore, PPyHA/JQ-1 effectively suppressed the expression of programmed cell death ligand 1 (PD-L1) and prolonged dendritic cell maturation and CD8+ T cell activation against the tumor both in vitro and in vivo. PPyHA/JQ-1 treatment simultaneously provides a significant tumor regression through photothermal therapy and immune checkpoint blockade, leading to a durable antitumor-immune response. Overall, our "Three-in-one" immunotherapeutic photo-activable nanoparticles have the potential to be beneficial for a targeted combinatorial treatment approach for TNBC. This article is protected by copyright. All rights reserved.