A method for preparing serotype O foot-and-mouth disease virus-like particles, the method including: construction of small ubiquitin-like modifier fusion expression vector, construction of recombinant expression vectors, construction of recombinant co-expression vector, expression and purification of proteins, and in-vitro assembly of serotype O foot-and-mouth disease virus-like particles. The disclosure also provides a test strip for detecting serotype O foot-and-mouth disease including a bottom board, and a detection layer disposed on the top of the bottom board. A detection line and a control line are disposed on the detection layer. An absorbent layer is disposed at one end of the detection layer close to the control line, and an immuno-gold pad is disposed at the other side of the detection layer close to the detection line. A sample pad is disposed on the top of the immuno-gold pad.