Graphene quantum dots (GQDs), which are edge-bound nanometer-size graphene pieces, have fascinating optical and electronic properties. These have been synthesized either by nanolithography or from starting materials such as graphene oxide (GO) by the chemical breakdown of their extended planar structure, both of which are multistep tedious processes. Here, we report that during the acid treatment and chemical exfoliation of traditional pitch-based carbon fibers, that are both cheap and commercially available, the stacked graphitic submicrometer domains of the fibers are easily broken down, leading to the creation of GQDs with different size distribution in scalable amounts. The as-produced GQDs, in the size range of 1-4 nm, show two-dimensional morphology, most of which present zigzag edge structure, and are 1-3 atomic layers thick. The photoluminescence of the GQDs can be tailored through varying the size of the GQDs by changing process parameters. Due to the luminescence stability, nanosecond lifetime, biocompatibility, low toxicity, and high water solubility, these GQDs are demonstrated to be excellent probes for high contrast bioimaging and biosensing applications. © 2012 American Chemical Society