Tofacitinib (TOF) is gaining recognition as a potent therapeutic agent for a variety of autoimmune disorders, including rheumatoid arthritis and psoriasis. Ensuring precise drug concentration control during treatment necessitates a rapid and sensitive detection method. This study introduces a novel electrochemical sensor employing a composite of nanodiamond (ND), copper aluminate spinel oxide (CuAl2O4), and iron (II, III) oxide (Fe3O4) as modified materials for efficient TOF detection. Extensive analyses using physicochemical and electrochemical techniques were carried out to characterize the morphological, structural, and electrochemical properties of the ND@CuAl2O4@Fe3O4 composite. Thereafter, various voltammetric methods were utilized to evaluate the electrochemical behavior of the ND@CuAl2O4@Fe3O4-modified glassy carbon electrode (GCE) concerning TOF determination. The fabricated electrode showcased superior performance in electrochemical TOF detection in a buffered solution (pH = 5), achieving a remarkably low detection limit of 7.8 nM and a linear response from 0.05 μM to 13.21 μM. Furthermore, applying the modified electrode as an electrochemical sensor exhibited exceptional selectivity, stability, and practicality in determining TOF in pharmaceutical and biological samples. Alongside the sensor development, this study conducted a thorough investigation using Density Functional Theory (DFT) for the geometry optimization of TOF and the TOF-ND complex. Consequently performed molecular docking studies using Janus Kinase 1 (JAK1) (PDB ID: 3EYG) and JAK3 (PDB ID: 3LXK) indicated higher interaction of the TOF-ND conjugate with the JAKs, reflected by binding energies of −12.9 kcal/mol and −11.7 kcal/mol for JAK1 and JAK3 respectively, compared to −7.0 kcal/mol and −6.9 kcal/mol for TOF alone. These findings illustrate the potential of the ND-based ND@CuAl2O4@Fe3O4 composite as a proficient sensing material for TOF detection and the merits of DFT in providing a detailed understanding of the interactions at play. This pioneering research holds promise for real-time TOF monitoring, which will advance personalized treatment strategies and improve therapeutic outcomes for patients with autoimmune disorders.