This research aimed to develop biomarkers for estimating ammonia (NH3) emissions from dairy cattle manure over a 15-day in vitro incubation system. To generate different levels of NH3 emissions, the experiment utilized four manure experimental groups: 1 urinary nitrogen (U) to 1 faecal nitrogen (F) ratio (CT), 2 U to 1 F ratio (2U1F), and CT and 2U1F with lignite application (CT + L and 2U1F + L, respectively). The addition of lignite to ruminant manure aimed to enhance environmental sustainability through its beneficial properties. Three biomarkers, nitrogen (N) isotopic fractionation (δ15N), N: potassium (K) ratio, and N: phosphorus (P) ratio, were investigated. Manure δ15N increased linearly when NH3 emission increased in CT and 2U1F groups (R2 = 0.79 and 0.90, respectively; P ≤ 0.001), while manure N: P decreased when NH3 emission increased in CT + L and 2U1F + L groups (R2 = 0.73 and 0.85, respectively; P ≤ 0.001). No useful relationship was found between N: K and NH3 emission, apart from in 2U1F group (R2 = 0.84; P ≤ 0.001). The experiment found manure δ15N and N: P are complementary biomarkers to predict NH3 emissions, from non-lignite and lignite groups, respectively.