Acute promyelocytic leukemia (APL) belongs to acute myeloid leukemia (AML) and is an M3 subtype in FAB typing. Some APL patients develop a promyelocytic leukemia/retinoic acid receptor fusion gene, the PML-RARα fusion gene. Under the combined action of various factors inside and outside, promyelocytic leukemia is onset. Embryonic stem cells (ESCs) have the ability to multi-differentiate. Under certain induction conditions, ESCs can differentiate into hematopoietic system. Promyelocytes are located downstream of ESCs differentiation and are a cell in the granulocyte differentiation stage. Exploring a non-labeled technical method to identify hematopoietic cells at different stages of differentiation has important scientific and practical significance. Raman spectroscopy can be used for differential diagnosis of many types of diseases, and its application prospects have become more extensive in recent years. This experiment investigated the Raman spectral characteristics of human embryonic stem cells leukemia cell line (ES), acute promyelocytic leukemia cell line (NB4) and leukemia cells from 4 patients with acute promyelocytic leukemia (M3), established a novel Raman label-free method to distinguish leukemia-related cells of distinct differentiation stages and provided basis for clinical research. Leukemia cells were collected from human embryonic stem cell line, acute promyelocytic leukemia cell line and bone marrow of patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 25~30 cells from each group or each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA), cluster analysis and partial least squares discrimination analysis (PLS-DA), and the spectra of three kinds of cells were analyzed and classified. Characteristics of Raman spectra were discussed combined with ultrastructure of leukemia cells. There were significant differences among Raman spectra of three kinds of leukemia-related cells. Compared with ES cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks which contributed to nucleic acids, proteins and lipids. Its biological mechanism involved the close relationship between APL and the PI3K/Akt/mTOR pathway. The PI3K/Akt/mTOR pathway was abnormally activated in acute promyelocytic leukemia cells, affecting the biomacromolecular metabolism of leukemia cells. The diagnostic models established by PCA-DFA, cluster analysis and PLS-DA could successfully classify these Raman spectra of different cells with a high accuracy of 100% (181/181). The model was evaluated by “Leave-one-out” cross-validation and reached a high accuracy of 98.9% (179/181). The proliferation and metabolism of M3 cells and NB4 cells were higher than those of ES cells. The diagnostic models established by PCA-DFA, cluster analysis and PLS-DA can classify these Raman spectra of different cells with a high accuracy. Raman spectra show consistent result with ultrastructure by TEM.