Humic substances (HS) are important soil components playing pivotal roles in guaranteeing long-term soil fertility. In this study, the chemical and biological properties of HS extracted from earthworm coprolites collected in soils subjected to different fertilization inputs (no fertilization, NF; fertilization with farmyard manure, FM; mineral input, M; mixed inputs, FMM, half farmyard manure plus half mineral input) were investigated. Results indicated a relationship between fertilization input and composition, molecular complexity and apparent molecular weight distribution of HS produced by earthworms. Coprolites from FM and FMM soils were the most enriched in organic carbon (OC), and HS from coprolites of FM soil were the highest in humic carbon (HC). Also, soil amendment with manure increased carboxylate and aromatic groups in HS, and the fraction with a high degree of polycondensation, thus indicating a positive impact of manure on plant residues’ degradation processes. These HS were the only to display hormone-like activity, which likely accounted for their most pronounced positive effects on plant growth and metabolism, including accumulation of chlorophylls, mineral nutrition, and activity of nitrogen assimilation enzymes, in oat (Avena sativa L.) plants growing in a soil-less system. We conclude that manure input favored the turnover of OC towards the humification process that led to the production of high-quality coprolites and HS with superior biological activity, and suggests that OC in coprolites and HC in HS from earthworms might be used as reliable indicators of soil fertility.