As a trace element nutrient, cobalt is critical for both prokaryotes and eukaryotes. In the current study, a turn-on Cobalt Bioluminescent Probe 1 (CBP-1) for the detection of cobalt has been successfully developed based on oxidative C-O bond cleavage. This probe exhibited high selectivity and sensitivity toward cobalt over other analytes. By using CBP-1, the successful in vivo imaging of cobalt accumulation was carried out in a mouse model. Such an ability to determine cobalt in living animals provides a powerful technology for studying the system distribution, toxic potency, and biological effect of Co2+.