The invention discloses a method for synchronously analyzing a base, a nucleotide, an organic acid, a fatty acid, an amino acid and a saccharide metabolic product with a two-step derivation method. The method comprises the following steps of: performing a series of physicochemical method technical treatment such as ultrasonic treatment, centrifugation, oximation, urea removing, phosphorus removing, sulfur removing, protein removing, nitrogen blowing, vacuum drying, and trimethyl silylation derivation on biological substrate samples (such as urea, blood, cerebrospinal fluid, and tissue fluid); and detecting the biological substrate samples by adopting a gas chromatograph-mass spectrum combination technology. Due to the adoption of method, programmed treatment can be performed on the biological substrate samples simultaneously, and over 400 kinds of metabolic intermediate and final products of over five kinds of substances can be detected at one time.