The invention provides a preparation method of a microfluidic monolithic column chip and an application of the chip in raman detection. The method comprises the steps that a polydimethylsiloxane (PDMS) microfluidic chip is prepared; a porous monolithic column solution is prepared; a monomer, a crosslinking agent, a pore-forming agent and a photoinitiator are subjected to ultrasonic mixing; nitrogen is supplied for deoxygenization; and finally the monolithic column solution is put in an ice bath for preservation. The solution is injected into a microfluidic chip pipeline, sealed and subjected to ultraviolet exposure, methanol washing is adopted for removing the pore-forming agent and the unreacted monomer, deionized water is used for washing sufficiently, a silver microsphere solution is injected into the microfluidic chip, ultra-sensitive raman real-time detection of a to-be-detected sample is achieved by utilizing silver microsphere enrichment and a surface raman enhancement effect, and a detection limit of the method can reach 10-12M. The method is convenient, flexible, low in cost, low in energy consumption, and convenient to popularize, and is widely applied to the fields of serum marker detection, gas marker detection, environmental monitoring, food safety and the like.