An 8‐week feeding experiment was conducted to determine the quantitative l‐lysine requirement of juvenile black sea bream Sparus macrocephalus (initial mean weight: 9.13 ± 0.09 g, SD) in eighteen 300‐L indoors flow‐through circular fibreglass tanks provided with sand‐filtered aerated seawater. The experimental diets contained six levels of l‐lysine ranging from 20.8 to 40.5 g kg−1 dry diet at about 4 g kg−1 increments. All the experiment diets were formulated to be isoenergetic and isonitrogenous. Each diet was assigned to triplicate groups of 20 fish in a completely randomized design. Weight gain and specific growth rate (SGR) increased with increasing levels of dietary lysine up to 32.5 g kg−1 (P < 0.05) and both showed a declining tendency thereafter. Feed efficiency ratio and protein efficiency ratio was poorer for fish fed the lower lysine level diets (P < 0.05) and showed no significant differences among other treatments (P > 0.05). All groups showed high survival (above 90%) and no significant differences were observed. The whole body crude protein and crude lipid contents were significantly affected (P < 0.05) by dietary lysine level, while moisture and ash showed no significant differences. The composition of muscle and liver also presented similar change tendency. Total essential amino acid and lysine contents in muscle both obtained the highest value when fish fed 32.5 g kg−1 lysine diet (P < 0.05). Serum protein, cholesterol and free lysine concentration were affected by different dietary treatments (P < 0.05), triacylglyceride and glucose contents were more variable and could not be related to dietary lysine levels. Dietary lysine level significantly affected condition factor and intraperitoneal fat ratio of juvenile black sea bream (P < 0.05) except for hepatosomatic index. There were no significant differences in white blood cell count and red blood cell count (P > 0.05), however, haemoglobin level was significantly influenced by different diets (P < 0.05). Analysis of dose (lysine level)‐response (SGR) with second order polynomial regression suggested the dietary lysine requirement of juvenile black sea bream to be 33.2 g kg−1 dry diet or 86.4 g lysine kg−1 protein.