Sea cucumbers can be explored as alternative raw materials by the pharmaceutical and fishery industries as anticancer agents because they contain potential bioactive compounds. This study aimed to determine the anticancer activity of Paracaudina australis extract against breast cancer cells (T47D) using an MTT assay. The secondary metabolites found in P.australis are steroids, terpenoids, saponins, and phenolics. The Thin-Layer Chromatography test results are indicated by Rf values, and steroid compounds in the ethyl acetate fraction are included in the standard Rf values. The isolates obtained were identified by GC-MS, FTIR, HPLC, and UV-V is spectrophotometry. The isolated compounds were Dihydrocholesterol and Cholestan-3-ol, with a molecular formula (C27H48O). The ion weight and molecular mass of the compound were m/z 388,7. This compound may be responsible for the anticancer activity of P.australis. The IC50 of Isolate F4 was 25,3 μg/ml, and IC50 of Isolate F7,8 was 13,76 μg/ml.