Phosphorite organic-rich sediments from Weng’an Doushantuo Formation preserve earliest animals and multicellular life, though it has been investigated by many researchers but in terms of biomarker analysis still need to be known. Here we have applied various specific aliphatic and aromatic biomarkers (Molecular fossils) parameters with stable carbon isotope data. The biomarker investigation of sixteen phosphoritic rocks from Weng’an Doushantuo Formation, Guizhou, South China, was carried out to identify the paleo-depositional environment, source of its organic matter and thermal maturity by using gas chromatography-mass spectrometry/ mass spectrometry (GC-MS). Particular care was taken while doing geochemical analysis in order to get rid of possible contaminations. Geochemical results indicate the source rock extracts from Weng’an Doushantuo Formation have similarity in molecular composition. All the studied samples have a lower amount of TOC, ranging from 0.07% to 0.58%. Molecular fossils revealed that the source input of organic matter was mainly from eukaryotic algae including dinoflagellates (or their ancestors) and demosponges as confirmed by steranes and terpanes. Pristane and phytane are derivative of Chlorophyll-a suggesting the availability of photosynthesis process in early ocean. Maturity based parameters suggest that the Weng’an phosphorite are highly mature, which is consistent with the thermal history of Doushantuo sediments. Isotopic compositions of δ¹³C_saturated and δ¹³C_aromatic hydrocarbon fractions show the marine environment. Biomarker investigation revealed that the phosphorite rocks from Weng’an Doushantuo Formation are highly mature, experienced strongly anoxic, clay rich conditions with major contribution of eukaryotic micro-organisms.