Extraction of short-chain fatty acids (SCFAs) from fermentation liquid of waste activated sludge (WAS) is the key bottleneck hindering its application as electron donor in denitrification. This study explores the feasibility of polyether-type polyurethane (PU)-modified layered double hydroxides (LDHs, prepared using eggshell waste as calcium source) in SCFAs adsorbing from WAS fermentation liquid (SFL). The adsorption parameters were first optimized by adsorption tests using artificial fermentation liquid (AFL). Then, adsorption kinetics, thermodynamic and isotherms were explored to further understand the adsorption mechanism. It revealed that SCFAs absorption by PU-LDHs from SFL was an endothermic and spontaneous process with positive enthalphy (ΔH) values and negative Gibbs free energy (ΔG) values. In addition, the maximum adsorption capacity of 208.0 mg SCFAs/g PU-LDHs was obtained from the Langmuir isotherm. Noting that both soluble carbohydrates and soluble proteins were simultaneously extracted, with efficiencies of 30.9%, 6.2%, respectively, compared with 62.9% SCFAs. The reuse tests confirmed that the prepared PU-LDHs can be used at least three times with high adsorptive capacity. With PU-LDHs-loaded SFL as external carbon source in the biodenitrification process, a denitrification rate of 0.014 mg NO-N/mg mixed liquid suspended solids (MLSS)·d was recorded. This study provided a sound basis for the preparation of cost-effective biodenitrification carbon source from SFL by a novel adsorbent.