The invention discloses a sample preparation and treatment method for researching the metaproteomics of soil. The method comprises the following steps: pretreating a soil sample to obtain a first suspension; carrying out concussion crushing to obtain a crushed first suspension; carrying out ultrasonic treatment, centrifuging the treated suspension, and collecting the obtained first supernatant; precipitating the first supernatant with cold acetone to obtain a protein solution; precisely weighing the protein solution containing proteins, adding a single protease or a protease combination, and incubating the proteins overnight to obtain a polypeptide solution; adding a separating agent 1, carrying out vortex treatment, adding a separating agent 2, carrying out vortex treatment, centrifuging the obtained solution, standing the centrifuged solution, and transferring about 90% of the obtained lower layer phase of a polypeptide-containing segment into a new centrifuging tube; adding a separating agent 3, carrying out vortex treatment to realize uniform mixing, centrifuging the treated solution, and taking the obtained lower layer; mixing the above obtained two peptide segment solution, desalting the obtained solution mixture, and collecting the obtained eluate; and centrifuging, concentrating and drying the eluate, afresh dissolving the obtained peptide segment sample, and directly injecting the dissolved sample to a machine for analysis. The method has the advantages of simplicity in operation, high stability, high recovery rate, completeness in extraction, and high flux, and is of great significance to massively develop the relevant researches of the metaproteomics of soil.