Circulating tumor cells (CTCs) are a promising diagnostic and prognostic biomarker for metastatic tumors. We demonstrate that CTCs’ diagnostic value might be increased through real-time monitoring of CTC dynamics. Using preclinical animal models of breast cancer and melanoma and in vivo flow cytometry with photoacoustic and fluorescence detection schematics, we show that CTC count does not always correlate with the primary tumor size. Individual analysis elucidated many cases where the highest level of CTCs was detected before the primary tumor starts progressing. This phenomenon could be attributed to aggressive tumors developing from cancer stem cells. Furthermore, real-time continuous monitoring of CTCs reveals that they occur at highly variable rates in a detection point over a period of time (e.g., a range of 0–54 CTCs per 5 min). These same fluctuations in CTC numbers were observed in vivo in epithelial and non-epithelial metastatic tumors, in different stages of tumor progression, and in different vessels. These temporal CTC fluctuations can explain false negative results of a one-time snapshot test in humans. Indeed, we observed wide variations in the number of CTCs in subsequent blood samples taken from the same metastatic melanoma patient, with some samples being CTC-free. If these phenomena are confirmed in our ongoing in vivo clinical trials, this could support a personalized strategy of CTC monitoring for cancer patients.