A method for preparing thermo-responsive iron-based nano-enzyme by reflux precipitation polymerization is disclosed. Isopropyl acrylamide or vinyl caprolactam and hydroxymethyl acrylamide are used asthe main monomers, reflux precipitation polymerization is adopted, and a simple reflux condensing tube is used to replace a complex distillation device and a solvent collection device. Then, a solventis refluxing in the system, magnetic stirring is also added to change dispersity. The method of the invention has higher efficiency, is simpler to operate and is more suitable for preparation of nanospheres. Then, phosphorus pentoxide is used for phosphate esterification of the composite microspheres, followed by chelation with Fe<2+> ions. Finally, the nano-catalysis microspheres with peroxidaseactivity are formed. The reaction is simple to operate, and the particles obtained have small and uniform particle size, large specific surface area and high catalytic activity.