The invention provides an isothermal nucleic acid amplification reaction reagent. The isothermal nucleic acid amplification reaction reagent is characterized by comprising components as follows: 100-800 mM of a Tris-HCl buffer solution, 10-150 mM of sodium chloride, 10-150 mM of potassium chloride, 10-50 mM of magnesium chloride, 5-15 mM of dithiothreitol, 5%-20% of polyvinylpyrrolidone, 10-20 mM of ATP (adenosine triphosphate), 1-5 mM of dNPTs, 10-50 mM of phosphoenolpyruvate, 500-1,500 ng/mu l of pyruvate kinase, 10-500 ng/mu l of BSA (bovine serum albumin), 25-200 pmol of each primer in a primer group, 50-200 ng/mu l of T4 bacteriophage DNA helicase gp41 protein, 100-500 ng/mu l of streptomyces coelicolor recA protein, 200-1,000 ng/mu l of single-strand binding protein and 50-200 ng/mu l of escherichia coli DNA polymerase I. The invention further provides an isothermal nucleic acid amplification method. According to the isothermal nucleic acid amplification reaction reagent and the isothermal nucleic acid amplification method, nucleic acid amplification under the isothermal condition at the lower temperature is realized, and a traditional nucleic acid amplification reaction process is greatly simplified.