Fluorescent carbon dots (FCDs) have received considerable attention because of the great potential for a wide range of applications, from bioimaging to optoelectronic devices. In this work, we reported the synthesis of nitrogen-doped FCDs with an average size of 2 nm in a subcritical water apparatus by using biomass waste (i.e., expired milk) as the precursor. The obtained FCDs were highly dispersed in aqueous solution because of the presence of O-containing functional groups on their surfaces. Under the excitation of ultraviolet and blue light, the FCDs exhibited excitation wavelength-dependent fluorescence in the emission range of 400-550 nm. The FCDs could be easily taken up by HeLa cells without additional surface functionalization, serving as fluorescent nanoprobes for bioimaging. The applications of FCDs as sensing agents for the detection of Fe3+, solid-state fluorescent patterning, and transparent hybrid films were also performed, demonstrating their potential for solid-state fluorescent sensing, security labeling, and wearable optoelectronics.