The invention discloses a preparation method and an application of pulse-invigorating and heart-nourishing pills. The pulse-invigorating and heart-nourishing pills consist of 100g of radix rehmanniae, 100g of caulis spatholobi, 60g of radix ophiopogonis, 60g of liquorice root, 60g of radix polygonum multiflorum preparata, 60g of colla corii asini, 60g of fruits of Chinese magnoliavine, 60g of radix codonopsis, 40g of turtle shell processed with vinegar, 40g of fructus jujubae and 20g of cassia twig. The pulse-invigorating and heart-nourishing pills are prepared by mainly crushing the liquorice root, the colla corii asini and the cassia twig, crushing the rest medicines and percolating the crushed medicines with 85% ethanol, collecting a percolate, decompressing and concentrating the percolate, drying the percolate, mixing an obtained material with the rest medicine powder and preparing the pills; or extracting the caulis spatholobi, the fruits of Chinese magnoliavine, the radix rehmanniae, the radix ophiopogonis, the radix codonopsis, the radix polygonum multiflorum preparata, the fructus jujubae and the turtle shell processed with vinegar twice with the addition of 70% ethanol, filtering an obtained extracting solution, decompressing and concentrating a filtrate, and crushing the liquorice root, the colla corii asini and the cassia twig; and mixing the materials and preparing the pills. The pulse-invigorating and heart-nourishing pills prepared by the method disclosed by the invention can significantly prolong the anoxia survival time of mice, showing an obvious anti-hypoxia effect.