Abstract: As an immunosuppressive agent isolated from Streptomyces, tacrolimus exerts its immunosuppressive effect mainly by interfering with T-cell activation. However, tacrolimus has a narrow therapeutic window, and excessively high concentrations can cause drug toxicity. Therefore, the degree of immunosuppression and adverse reactions of tacrolimus are closely related to its blood concentration, even when the blood concentration is within the therapeutic window, patients often experience adverse reactions such as nephrotoxicity and digestive system discomfort. Among various detection methods for blood immunosuppressants, mass spectrometry has become the gold standard for clinical therapeutic drug monitoring (TDM) of immunosuppressants. Compared with traditional ionization technologies, paper spray technology has significant advantages, as it can greatly reduce the use of solvents, simplify pretreatment steps, and improve analysis efficiency. In this study, a new multistage fragmentation method for the rapid detection of tacrolimus in plasma by paper spray in-situ mass spectrometry was established using an independently developed quadrupole-linear ion trap tandem mass spectrometry system. To minimize detection time, plasma samples were precipitated with an organic solvent containing an internal standard, and then the supernatant was aspirated for detection. Due to the complexity of the sample matrix, the tertiary fragment ions of tacrolimus were used for quantitative analysis. By optimizing the conditions of the paper spray ion source, such as the type of paper substrate, elution solvents, shape of the paper, distance between the paper tip and the mass spectrometer port, spray voltage, as well as the mass spectrometry conditions, the detection sensitivity and limit of quantification were significantly improved. The results demonstrated a good linear relationship for tacrolimus within the linear range of 5.00 to 1 000.00 μg/L, with a correlation coefficient (R²) of 0.998. The limit of detection and limit of quantitation are 1.00 and 5.00 μg/L, respectively. The accuracy ranges from 95.23% to 102.39%, with intra-day and inter-day precision ranging from 6.17% to 11.90% and 5.30% to 13.39%, respectively. The matrix effect normalization factor (RSD) is less than 8.88%. This method was applied to monitor tacrolimus levels in 20 plasma samples, with concentrations ranging from 5.62 to 36.75 μg/L and RSD between 1.75% and 14.41%, indicating that the method has good precision and can meet the needs of clinical blood drug concentration monitoring. The entire detection process only takes 1 min to obtain quantitative results, and in scenarios requiring rapid detection of blood drugs, this method is expected to become an efficient alternative, with the potential to be applied to the concentration monitoring of other immunosuppressants.