Free fatty acids (FFAs) are the important material used in food, personal care, emulsifiers, adhesives and surfactants. In order to enhance the preparation of FFAs, the effects of reaction variables, optimization, thermodynamic property for the Amano lipase PS catalyzed hydrolysis of pine nut oil (PNO) using deep eutectic solvents (DESs) as co-solvents were studied. The results showed that FFAs could be successfully prepared from pine nut oil through Amano lipase PS catalyzed hydrolysis using Choline chloride:Urea (ChCl:U, 1:2, mol/mol) as co-solvent. Under the optimal conditions (reaction temperature 46°C, water amount 38%, DES addition 43%, lipase dosage 7.6%, reaction time 13 h), the maximum content of FFAs in the products and degree of hydrolysis (DH) of oil were up to 89.1 ± 1.9% and 92.7 ± 2.2%, respectively. The effects of reaction variables on the hydrolysis increased in the order of DES addition < reaction temperature < reaction time < lipase dosage < water amount. The thermodynamics (Arrhenius equation) for the triglycerides hydrolysis was V = 4289.39·exp(-22942.09/RT) with the activation energy (Ea) of 22.94 kJ/mol. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were 81.50 ± 2.64 kJ/mol, 20.18 ± 0.12 kJ/mol and -184.59 ± 0.36 J/mol/K, respectively. The lipase in the aqueous DES could be directly re-used for 3 times.