To provide reference values of trans-laminar cribrosa pressure difference (TLCPD) and reveal the association of TLCPD with systemic biometric factors.In this cross-sectional study, 526 quasi-healthy subjects (including 776 eyes) who required lumbar puncture for medical reasons were selected from 4915 neurology inpatients from 2019 to 2022. Patients with any diseases affecting intraocular pressure (IOP) or intracranial pressure (ICP) were excluded. The ICPs of all subjects were obtained by lumbar puncture in the left lateral decubitus position. IOP was measured in the seated position by a handheld iCare tonometer prior to lumbar puncture. TLCPD was calculated by subtracting ICP from IOP. Systemic biometric factors were assessed within 1 h prior to TLCPD measurement.The TLCPD (mean ± standard deviation) was 4.4 ± 3.6 mmHg, and the 95% reference interval (defined as the 2.5th-97.5th percentiles) of TLCPD was -2.27 to 11.94 mmHg. The 95% reference intervals for IOP and ICP were 10-21 and 6.25-15.44 mmHg, respectively. IOP was correlated with ICP (r = 0.126, p < 0.001). TLCPD was significantly negatively correlated with body mass index (r = -0.086, p = 0.049), whereas it was not associated with age, gender, height, weight, blood pressure, pulse, or waist and hip circumference.This study provides reference values of TLCPD and establishes clinically applicable reference intervals for normal TLCPD. Based on association analysis, TLCPD is higher in people with lower BMI.© 2022. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.