Liquid-liquid phase separation (LLPS) is a reversible process, during which biological macromolecules, including proteins and nucleic acids, condense into liquid membraneless organelles under the influence of weak multivalent interactions. Currently, fluorescence recovery after photobleaching is the primary method used to detect the phase separation of biological macromolecules. Recent studies have revealed the link between abnormal LLPS and the pathogenesis and development of various human cancers. Through phase separation or abnormal phase separation, tumor-related biological macromolecules, such as mRNA, long noncoding RNAs (lncRNAs), and tumor-related proteins, can affect transcriptional translation and DNA damage repair, regulate the autophagy and ferroptosis functions of cells, and thus regulate the development of various tumors. In this review, we summarized the latest research findings on the mechanism of LLPS in the pathogenesis and progression of tumors and elaborated on the promotion or inhibition of autophagy, tumor immunity, DNA damage repair, and cell ferroptosis after abnormal phase separation of biomolecules, including mRNA, lncRNA, and proteins, which subsequently affects the pathogenesis and progression of tumors. According to published findings, many biological macromolecules can regulate transcriptional translation, expression, post-transcriptional modification, cell signal transduction, and other biological processes through phase separation. Therefore, further expansion of the research field of phase separation and in-depth investigation of its molecular mechanisms and regulatory processes hold extensive research potential.