The invention provides a tracheotomy tube and puncture tube assembly. The assembly comprises a scalpel, an injector, a guiding guide wire, a puncture trocar, a short dilator, a guiding catheter, a single-stage dilation tube, a tracheotomy tube guiding tube and a tracheotomy tube. The invention also provides a using method for the assembly and a storage box. According to the tracheotomy tube and puncture tube assembly, preoperative preparation time is shortened, the preoperative preparation is more accurate, convenient and efficient, unnecessary incision injury of the traditional operation can be avoided, wounds and bleeding are reduced, the wounds are well fitted to instruments, and the nursing difficulty is effectively reduced; when the assembly is adopted for intubation, the wounds are good in resilience and left scars are smaller. The storage box is used for storing the tracheotomy tube and puncture tube assembly, so that the tracheotomy tube and puncture tube assembly is arranged in order and easy to store, take and transport.