PET myocardial perfusion imaging (MPI) is the gold standard for the noninvasive diagnosis of ischemic myocardial. Construction of 18F-labeled PET MPI probe showed benefits to reduce the imaging cost, and enhance the image quality and patient-friendliness. Two 18F-labeled MPI probes (18F-BoMPI) were developed. Detailed in vitro/vivo evaluation including photophysical properties, in vitro stability, myocardial cell uptake kinetics and mechanisms, cytotoxicity and IC50, biodistribution and plasma clearance curve were investigated. Resting and stressing myocardial perfusion PET imaging were performed in healthy and myocardial ischemic mice. 18F-BoMPI could be quickly labeled and easily postprocessed, and demonstrated excellent in vitro stability. Cell assays indicated that 18F-BoMPI exhibited mitochondria-targeting but potential-independent myocardial uptake. In vivo evaluation revealed the effective myocardial uptake and rapid background clearance. PET MPI confirmed effective probe accumulation in the healthy heart, but rapidly clearance in the background, making heart clearly delineated in the images. Ischemic myocardial could be clearly distinguished as the region of radioactivity sparsity in PET MPI. The 18F-labeled probes showed great potentials to reduce the practicability threshold of PET MPI.