In a Kolthoff buffer solution (pH = 10.7), the fluorescence intensity of acridine red, which is quenched by Pd²⁺, can be enhanced by adding a certain amount of laevorotatory-isoleucine (L-isoleucine). Therefore, we have established a fluorescence spectrometry method for the detection of L-isoleucine. When the excitation slit width is 5 nm and emission slit width is 10 nm, we obtain the following results: the linear regression equation: F = −33.884 + 468.07C (mg L⁻¹), the correlation coefficient: R = 0.9984, linear range: 0.125–1.375 mg L⁻¹ and the detection limit: 0.00027 mg L⁻¹. Furthermore, we calculate the association constants of Pd²⁺–acridine red (k₁ = 1.62 × 10²) and Pd²⁺–isoleucine (k₂ = 7.04 × 10²) using a spectrophotometric method.