Carbon dots (CDs), with high photostability, tunable excitation and emission wavelength, excellent biocompatibility, and environmental friendliness, attract great interest because of their promising applications in various fields. The synthesis of CDs from renewable biomass is especially attractive due to its sustainable and cost-efficient feature. However, one of the major problems for this route is the low yield of CDs. To overcome this issue, we propose a facile, universal, and highly effective synthesis method to prepare fluorescent CDs from various biomass hydrothermal carbons (HTCs). As compared with the current methods for synthesizing biomass derived CDs, our approach offers ultrahigh yield (42.5% based on the weight of biomass and 99.0% based on the weight of HTC) that is much higher than those obtained from other methods. The physiochemical properties of the as-prepared CDs can be tuned by reaction temperature. Furthermore, the PL quantum yield is high up to 16.6%, and is correlated with the surface chemical groups and conjugated π-domains of the carbon core in CDs. The fluorescence of CDs is effectively and selectively quenched by Cu2+, which enables CDs applying as fluorescent Cu2+ nanoprobe with a linear range of 0–30 μmol L−1 and a detection limit of 85 nmol L−1. Due to simple operation, mild condition, and low cost of the process, this method have a great potential in the large-scale synthesis and application of CDs from biomass.