By application of preparative high-speed counter-current chromatography (HSCCC) to the crude quinolone alkaloids (1.1 g) from the fruit of Tetradium ruticarpum, 1-methyl-2((6Z,9Z)-pentadecadienyl)-4(1H)-quinolone (1, 8.4 mg), dihydroevocarpine (2, 27.0 mg), and 1-methyl-2-pentadecyl-4(1H)-quinolone (3, 18.8 mg) were isolated in one step with sufficient purity using the solvent system composed of hexane-ethyl acetate-methanol-water (Hex-EtOAc-MeOH-H2O, 5: 2: 5: 3). Further purification of the subfraction was performed by amending the solvent composition and achieved another three quinolone alkaloids, i.e., 1-methyl-2-undecylquinolin-4(1H)-one (4, 13.7 mg), (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one (5, 14.0 mg) from subfraction FR3-A3-85 using Hex-EtOAc-MeOH-H2O (5:3.5:8.75:8.25), and 1-methyl-2-nonylquinolin-4(1H)-one (6, 15.1 mg) from subfraction FR3-A3-36 using Hex-EtOAc-MeOH-H2O (5:3.8:5:4.8). The relationship between the structure of the six alkaloids and their affinities for bovine serum albumin (BSA) was investigated using fluorescence titration analysis. The length and the presence of double bond of the side chain affected their binding process with BSA. The binding behavior might influence their other biological activities.