The invention discloses a novel method for measuring hydrogen pumping capability of mitochondria, and belongs to the field of scientific research and experiments. The method includes S1) putting a magnetic stirrer below an absorption cell rack of a spectrophotometer; S2) setting the spectrophotometer into a time scanning state; S3) adding an absorption cell into the spectrophotometer, adding KCl into the absorption cell, starting the magnetic stirrer and starting scanning; S4) adding 8-hydroxy-1,3,6-pyrenetrisulfonic acid; S5) adding ferrocytochrome c, valinomycin, 2,3-dimethoxy-5-methyl-6-(10-bromo)-1,4-benzoquinone and a mitochondrion solution; S6) recording an absorption value A1 after an absorption line is flat; S7) adding 1 [mu]L of sodium ferricyanide having a concentration of 5 mM,and recording an absorption value A2; S8) adding a proton carrier that is [(3-chlorophenyl)hydrazono]malononitrile, and recording an absorption value A3; and S9) adding sodium ferricyanide, and recording an absorption value A4; and S10) calculating the hydrogen pumping capability according to an equation that is (H<+>/e)=x/y=(A1-A2)/(A3-A4). The method is reasonable in design, simple to operate and low in interference by the environment, and a measurement result is good in precision and repeatability. The method is suitable for use in common laboratories.