Objective: To analyze the application and testing of non-surgical routine blood test items in clinical diagnosis and treatment in a top-tier ophthalmic hospital over the past five years, providing ophthalmologists with insights into the overview of laboratory-assisted diagnosis, laboratory project management in specialized hospitals, and the basis for launching new projects. Methods: Relevant test records of 22,453 samples (totaling 94,081 test items) from 12,866 outpatient patients who applied for blood tests at the Zhongshan Ophthalmic Center of Sun Yat-sen University from January 1, 2018, to December 31, 2022, were retrieved from the medical laboratory information management system. The departments applying for tests and specialist physicians were divided into 10 subspecialties according to the recommendations of the Ophthalmology Branch of the Chinese Medical Association. Statistical analysis was performed on disease diagnosis and test items, and the results were described in the form of text, bar graphs, and line graphs. Results: Among the 12,866 outpatient patients who applied for blood tests over the five-year period, 6,356 (49.4%) were male and 6,510 (50.6%) were female. Based on the classification of first-visit ophthalmic diseases, the top three were ophthalmic plastic surgery/ocular tumor diseases (5,214 cases, 40.5%), fundus diseases (3,487 cases, 27.1%), and corneal diseases (1,711 cases, 13.3%). The number of samples applied for testing increased from 3,163 in 2018 to 5,903 in 2022, showing an overall upward trend. From the perspective of specialist physicians applying for tests, ophthalmic plastic surgery/orbital disease specialists had the highest number of applications, with 6,751 (30.1%), among which autoimmune disease testing accounted for the highest proportion, at 49.1%, and thyroid disease-related testing accounted for 41.9%. The number of applications from ophthalmic immunology specialists was 4,214 (18.8%), with autoimmune disease testing accounting for 55.7% and infectious disease testing accounting for 32.5%. The number of applications from fundus disease specialists was 3,629 (16.2%), with autoimmune disease testing and infectious disease testing accounting for 47.8% and 39.6%, respectively. The number of applications from corneal disease specialists was 1,436 (6.4%), with allergic disease testing accounting for 41.2%. Since multiple tests could be performed on a single application, the ophthalmic plastic surgery/orbital disease subspecialty had the highest total number of tests applied for, with 33,513 tests, of which thyroid disease testing accounted for 65.0%. The corneal disease subspecialty applied for 16,482 tests, with allergic disease testing accounting for 83.4%. The fundus disease subspecialty and ophthalmic immunology subspecialty respectively applied for 8,794 and 8,047 tests, both primarily focused on autoimmune disease testing, accounting for 42.5% and 40.4%, respectively. Conclusions: The number of applications for non-surgical routine test items in ophthalmic specialized hospitals is significantly unevenly distributed among various subspecialties, and the composition of the items is influenced by the characteristics of diseases in each subspecialty. Ophthalmic laboratories should strengthen the promotion and management of non-surgical routine test items in a targeted manner.