Photothermal biosensors with the advantages of speed and high sensitivity offer alternative and reliable solutions for real-time clinical diagnosis, food safety testing and environmental monitoring. Although metallic nanoparticles are usually used for photothermal biosensors, their poor photothermal stability and potential toxicity hinder clinical applications. Taking advantage of the low cytotoxicity and remarkable photothermal effect under low laser power of polypyrrole-based organic nanoparticles, we developed a novel photothermal biosensor with temperature and pressure dual readout. After the formation of immunoassay sandwich structures, polypyrrole as the photothermal agent is synthesized in-situ with pyrrole, HCl and Fe3+ released from magnetic Fe2O3 particles modified with detection antibody. The heterocyclic rings from polypyrrole enable photothermal performance in the NIR region. The resulting increased heat and pressure in a sealed well are measured by a digital thermometer and a portable pressure meter, respectively. Taking C-reactive protein (CRP) as a model target, the proposed strategy allowed sensitive, selective and accurate analysis of biomarkers, and showed performance comparable to that of ELISA. Overall, the dual-mode photothermal biosensor holds great potential for simple and low-cost photothermal sensing of biomarkers for point-of-care testing (POCT).