The invention discloses a chloroplast complex V activity determination kit based on a micro-method and a method. The kit comprises a reagent 1 (Tris-HCl, sucrose, EGTA), a reagent 2 (ATP), a reagent 3(Tris-HCl, MgCl2, KCl, NaCl), a reagent 4 (TCA), reagent 5 (ASA), a reagent 6 (ammonium molybdate), and a reagent 7 (sulfuric acid, water). The method including extracting and detecting activity of chloroplast complex V is established for the first time, the principle is that complex V hydrolyzes ATP to produce ADP and Pi, and activity of complex V is determined by measuring the increasing rate of Pi; by reasonably arranging a contrast tube, the influence of impurities can be eliminated, and the detection specificity is very high; the method can effectively separate cytoplasmic protein, mitochondrial protein and chloroplast protein, and accurately measures the activity of complex V located in chloroplast; and the method has good repeatability, and the coefficient of variation is within 2%.