The aim of this study was to identify key markers of minimal residual disease (MRD) in childhood Acute Lymphoblastic Leukemia (ALL). Bone marrow samples were collected at presentation from 139 patients with newly diagnosed B-lineage ALL. On the basis of the expression of CD19, CD10, and CD34 antigens by bone marrow cells, combined with the terminal deoxynucleotide transferase (TdT), CD38, CD45, CD58, CD21, CD66c, CD22, and CD33 expression patterns characterized at diagnosis, leukemia-associated immunophenotypes (LAIPs) were identified. One hundred thirty-nine patients with a median age of 4.3 years were screened with 4-color flow cytometry MRD screening, and 119 of them exhibited 1 or more LAIP suitable for further monitoring, constituting a coverage rate of 85.6%. Only 20 of the 139 (14.4%) had no LAIP identified for follow-up. The most applicable antibody combination was TdT/CD10/CD34/CD19 (87/139, 62.6%), followed by CD38/CD10/CD34/CD19 (85/139, 61.2%) and CD45/CD10/CD34/CD19 (58/139, 41.7%). We have identified a relatively effective MRD panel, combined with TdT, CD38, and CD45 as key markers, that is applicable to the majority of newly diagnosed B-lineage ALL.