1. The aim of this study was to evaluate the tolerance of laying hens for an excessive L-valine (L-val) supply on laying performance, egg quality, serum free amino acids, immune function and antioxidant enzyme activities of laying hens. 2. A total of 720 HyLine Brown hens were allocated to 5 dietary treatment groups, each of which included 6 replicates of 24 hens, from 40 to 47 weeks of age. Graded amounts of L-val were added to the basal diet to achieve concentrations of 0 (control), 1, 2, 3 and 4 g/kg, respectively, in the experimental diets. 3. Supplementing the diet with L-val did not affect egg production, egg mass, egg weight, feed conversion ratio (FCR) or egg quality. The average daily feed intake response to supplemental L-val was quadratic and was maximised at 2.0 g L-val/kg diet. No differences were observed for total protein, total amino acids, blood urea nitrogen (BUN), uric acid, lactate dehydrogenase (LDH), alkaline phosphatase (AKP), Ca and P concentrations among the treatments. 4. Serum albumin concentration increased significantly in response to supplemental L-val and was also maximised at 2.0 g/kg. In addition, serum glucose increased quadratically to peak at 2.0 g L-val/kg diet. Serum free valine increased as L-val concentration increased to 2.0 g/kg diet and then decreased linearly. 5. Supplementation of L-val did not affect the serum concentrations of total antioxidative capability (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA). L-val supplementation did not affect the concentrations of immunoglobulins IgG, IgA, IgM and complements (C3 and C4). Serum concentration of triiodothyronine (T3) increased significantly at 2.0 g L-val/kg diet. 6. It is concluded that high concentrations of L-val are tolerated and can be successfully supplemented into diets without detrimental effects on laying performance or immune function of laying hens.