Despite their efficiency and specificity, the instability of natural enzymes in harsh conditions has inspired researchers to replace them with nanomaterials. In the present study, extracted hemoglobin from blood biowastes was hydrothermally converted to catalytically active carbon nanoparticles (BDNPs). Their application as nanozymes for the colorimetric biosensing of H2O2and glucose and selective cancer cell-killing ability was demonstrated. Particles that were prepared at 100 °C (BDNP-100) showed the highest peroxidase mimetic activity, with Michaelis–Menten constants (Km) of 11.8 mM and 0.121 mM and maximum reaction rates (Vmax) of 8.56 × 10−8mol L−1s−1and 0.538 × 10−8mol L−1s−1, for H2O2and TMB, respectively. The cascade catalytic reactions, catalyzed by glucose oxidase and BDNP-100, served as the basis for the sensitive and selective colorimetric glucose determination. A linear range of 50–700 µM, a response time of 4 min, a limit of detection (3σ/N) of 40 µM, and a limit of quantification (10σ/N) of 134 µM was achieved. In addition, the reactive oxygen species (ROS)-generating ability of BDNP-100 was employed for evaluating its potential in cancer therapy. Human breast cancer cells (MCF-7), in the forms of monolayer cell cultures and 3D spheroids, were studied by MTT, apoptosis, and ROS assays. The in vitro cellular experiments showed dose-dependent cytotoxicity of BDNP-100 toward MCF-7 cells in the presence of 50 µM of exogenous H2O2. However, no obvious damage was induced to normal cells in the same experimental conditions, verifying the selective cancer cell-killing ability of BDNP-100.