Tacrolimus (TAC) is a first-line immunosuppressive agent in clinical practice, with therapeutic decisions currently guided primarily by whole-blood concentration monitoring. However, with the deepening of research and clinical practice, it has been indicated that patients may experience suboptimal efficacy or toxic reactions even when whole-blood concentrations remain within the therapeutic window, rendering current monitoring strategies inadequate for clinical needs. TAC primarily acts within lymphocytes, making intracellular TAC concentration a more accurate indicator of its pharmacodynamic activity and a key driver for individualized treatment. Research on intracellular TAC levels remains in its early stages. Clarifying the relationship between intracellular TAC concentrations and clinical efficacy, identifying relevant influencing factors, and understanding the limitations of current detection technologies are crucial for promoting its standardized clinical application. Based on the pharmacokinetics and pharmacodynamics of TAC, this review summarizes the research progress on intracellular TAC concentrations domestically and internationally, aiming to provide insights for optimizing therapeutic drug monitoring of TAC.