Real-time ultrasound imaging (US) to measure abdominal muscle dimensions has aided low back pain rehabilitation and research. Notwithstanding, ultrasound imaging measurement of transversus abdominis muscle activation in chronic low back pain populations has been characterized by variable and generally suboptimal intra-observer reliability. Methodological deficiencies of ‘freehand’ ultrasound imaging are uncontrolled probe–skin pressure, inclination and roll of the probe. Despite previous attempts to standardize these parameters, intra-observer reliability in chronic low back pain was poor to moderate (0.32–0.62). Therefore, a standardized method that controls and records probe force, inclination and roll during ultrasound imaging may optimize measurement reliability in chronic low back pain. This pilot study investigated utility, standardization and intra-observer reliability of ultrasound imaging transversus abdominis thickness measurement in chronic low back pain patients (n = 17). Transversus abdominis imaging over two separate measurement sessions was conducted using a novel method to standardize probe parameters. Resting and contracted transversus abdominis thickness, and transversus abdominis activation measurements were obtained from duplicate paired images (n = 68). Intra-class correlation coefficients were reported with 95% confidence intervals. Transversus abdominis thickness at rest (intra-class correlation coefficient = 0.97 confidence interval: 0.93, 0.99), when contracted (intra-class correlation coefficient = 0.99 confidence interval: 0.97, 0.99) and transversus abdominis activation (intra-class correlation coefficient = 0.93 confidence interval: 0.81, 0.97) measurements were highly reliable. Ultrasound imaging of transversus abdominis using the novel standardized ultrasound imaging method produced highly reliable intra-observer transversus abdominis measurements, superior to ‘freehand’ ultrasound imaging, despite the physical limitations typically associated with a chronic low back pain population. Unique standardizing ranges for ‘probe force device’ probe parameters were obtained. This novel standardized ultrasound imaging method may optimize transversus abdominis activation assessment in chronic low back pain and other populations, aiding future research.