Glycolysis, as a multi-step oxidation process, plays important roles in the energy supply for living cells, including malignant tumor cells. Recent studies have revealed that 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (named PFKFB3), a bifunctional enzyme in glycolysis, is upregulated in a variety of malignant solid tumors and has been regarded as a potential biomarker for the diagnosis and treatment of tumor patients. Based on the structure of selective PFKFB3 inhibitors, we designed and synthesized a radio-metal radiolabeled small molecule, Ga-68-5, which also showed potent selectivity in enzymatic and biochemical tests (with an IC50 value of 12.5 nM). According to further in vitro and in vivo evaluations, Ga-68-5 showed promising properties as a PET ligand, and selective accumulation in PFKFB3-positive tumors was observed in PET images (with max SUV values of 0.60). Our results indicated that radio-metal radiolabeled aminoquinoxaline derivative, as represented by Ga-68-5, held the potential to be developed as selective PFKFB3-targeted PET tracers, and further investigation and optimization would also be required for this scaffold.