The invention provides a chemiluminescent substrate solution, which comprises the following components: 0.2-1.2g/L of Luminol, 0.1-5g/L of cinnamic acid, 0.05-0.6g/L of 4-iodophenylboronic acid, 0.05-0.6 g/L of p-iodophenol, 5-50ml/L of dimethylformamide (DMF), 2.5-7.5 g/L of polyvinyl alcohol (PVA), 5-15g/L of polyvinyl pyrrolidone (PVP), 1.5-5g/L of polyethylene glycol 600 (PEC -600), 1-10g/L of ethylene diamine tetraacetic acid (EDTA), 400,000-4,000,000 unit/L of gentamicin sulfate, 0.1-1.0g/L of carbamide peroxide and 0.1mol/L of tris buffer solution with a pH vale being 9.0. The chemiluminescent substrate solution is a solution which can be directly used, and maintains wide linear detection range, good stability and high sensitivity, thus solving the problem that a traditional chemiluminescent substrate solution consists of a solution A and a solution B which are stored separately; and after the solution A and the solution B are isovolumetrically mixed for 10 minutes, signal values are decreased sharply, the detection range is narrowed and the stability is poor. In addition, the chemiluminescent substrate solution provided by the invention is easy to prepare, low in cost and simple to operate, environmentally friendly, and non-toxic and harmless to human bodies and the like.