The invention discloses a recombinant expression cassette for efficiently expressing soluble oxalic acid oxidase and a preparation method of an oxalic acid oxidase capable of efficiently expressing solubility and activity in Escherichia coli. Cellular supernatant is precipitated through ammonium sulfate, and a solution, which is re-dissolved through citric acid-sodium-citrate and contains oxalic acid oxidase, is directly applied to activity determination of oxalate decarboxylase. Compared with traditional methods such as commercially available oxalate determination kits and HPLC (high performance liquid chromatography), the determination method is higher in accuracy and sensitivity, low in time consumption and cost, applicable to high-throughput determination on a large number of samples through a micro-plate reader and high in practicality. The prepared oxalic acid oxidase can be applied to activity determination of enzymes with oxalic acid as substrate or product as well as to determination of the content of the oxalic acid in blood or urine of human or animals as well as various samples containing oxalic acid.