In this report, a facile one-step synthesis strategy has been developed for producing fluorescent carbon nanospheres (CNs) using lactobionic acid (LBA) as a precursor. The resulting CNs are highly stable in aqueous solution with an average size of 120 nm. The obtained CNs contained large amounts of –OH and –COOH on their surfaces which will facilitate further functionalization for more additional usage. In addition, carbon nanospheres possess an intrinsic fluorescence property so they can be used for fluorescence imaging. Our CNs exhibited remarkable photoluminescence properties and low cytotoxicity which can be used to label living cells with high efficiency, suggesting potential applications in biolabeling and bioimaging. In brief, we developed novel fluorescent carbon nanospheres with high stability, biocompatibility and labeling efficiency for cell imaging, as well as abundant active groups on their surface for further modifications.