The invention discloses a kit and a method for measuring the content of 1,3-diphosphoglycerate based on a micromethod. A 
reagent of the kit comprises an extracting solution, a first 
reagent and a second 
reagent, wherein the extracting solution is prepared from 
Tris-HCl, EGTA, mercaptoethanol and glycerinum in a 
mixed mode, the first reagent is prepared from MgSO4.7H2O, NADH, ATP and 
glyceraldehyde-3-
phosphate dehydrogenase in a 
mixed mode, and the second reagent is prepared from 
Tris-HCl. The method comprises the principle that the 
glyceraldehyde-3-
phosphate dehydrogenase can reversely catalyze the 1,3-diphosphoglycerate and the NADH to generate 
glyceraldehyde-3-
phosphate, 
inorganic phosphorus and NAD, the glyceraldehyde-3-phosphate 
dehydrogenase and the NADH are added into a 
reaction system, and by measuring the reduction amount of the NADH at 340 nm, the content of the 1,3-diphosphoglycerate can be reflected. The kit and the method for measuring the content of the 1,3-diphosphoglycerate have the characteristics that the application range is wide, operation is easy and convenient, the detection time is short, detection sensitivity is high, the cost is low, 
repeatability is good, and the accuracy rate is high.