The invention discloses a method for determining the minimal residual lesion level based on multiple amplification sequencing. Comprising the following steps: S1, sample treatment, S2, primer pool manufacturing, S3, single-ended amplification, S4, quality control, S5, purification, S6, mixed sequencing, S7, sequencing data analysis, and S8, minimal residual lesion level calculation. According to the determination method, simple and convenient single-ended amplification is used, the minimal residual lesion level can be accurately and quantitatively detected, an external reference is not needed, meanwhile, primer dimers can be prevented from being generated, the proportion of effective data is increased, and then the accuracy of determining the MRD level is improved.