Novel, uniform, and ordered biomass carbon materials using the skin tissue of tamarind seedcase (TS) as the carbon source were first synthesized by one-step carbonization or hydrothermal carbonization (HTC) without being template-assisted and are further explored for highly sensitive H 2 O 2 biosensing. The results demonstrated that the obtained biomass carbonaceous materials (TS-C, TS powder (TSP), TSP-C, TS-HTC, and TSP-HTC) with obviously different morphologies, absorption characteristics, and pore size distributions showed variable electrocatalytic properties. TS-HTC with promising pitaya flower-like structures and a high specific surface area of 436.425 m 2 g −1 presented the optimized catalytic performance for H 2 O 2 detecting at the peak potential of −0.38 V. The corresponding H 2 O 2 sensor displayed an outstanding sensitivity of 122.02 μA mM −1 cm −2 at the H 2 O 2 concentration range of 0.05−0.5 mM and 80.79 μA mM −1 cm −2 (0.5−2.7 mM), respectively. The response time was short at 3.61 s, indicating that TS-HTC has turned out to be an attractive material for designing H 2 O 2 biosensors, with a promising internally interlinked reticulate-like structure for capturing biomolecules. Moreover, the direct detection of H 2 O 2 released from Hep3B cells is demonstrated on TS-HTC-based sensors, being effective to detect H 2 O 2 activity in situ for the early diagnosis of liver cancer, suggesting its potential application for efficient recognition and determination of H 2 O 2 in fields of life science, clinics, disease prevention, and so on.