A novel, accurate, precise, inexpensive, and sensitive spectrophotometric approach is established to assay levodopa (LD) in both bulk drug and its pharmaceutical formulations. The method depends on the reaction of the ternary complex of a mixed-ligand type among LD, nickel(II) (Ni(II)), and 2,3-diaminopyridine (DAP) to form a stable complex of Ni-LD-DAP which is not extractable. The complex showed maximum absorption at 478 nm, with the apparent molar absorptivity of 4.93 × 103 L·mol−1·cm−1 and Sandell’s sensitivity of 0.04 μg·cm−2. Beer’s law is obeyed within the concentration range of 2–58 μg·mL-1, and the regression line equation is as follows: Y = 0.0251X + 0.0045 (R2 = 0.9990; n = 5). The detection limit and quantitation limit are found to be 0.1388 μg·mL−1 and 0.4207 μg·mL−1, respectively. The present study achieved reasonable accuracy (average recovery of 100.17%) and good precision (RSD did not exceed 1.71%). The developed method has been validated according to the current ICH guidelines (2013). The results of pure LD and pharmaceutical preparations using the recommended spectrophotometry are promising.