Abstract: Maintenance of acid–base homeostasis is critical in human tissues. Many pathological processes, such as ischemia, renal failure, inflammation, cancer, and chronic obstructive pulmonary disease, involve in acid–base imbalance (pH alteration). Acidic tumor pH can be used as a target and universal biomarker for cancer imaging and therapy. Magnetic resonance molecular imaging (MRMI), nuclear medicine imaging, and optical imaging have been used in preclinical imaging of acidic tumor pH and also used in clinical investigation. However, currently non-invasive molecular imaging was unavailable in routine clinic imaging of tumor pH. The molecular imaging of acidic tumor pH was summarized in this review.