To compare the accuracy of transvaginal ultrasound (TVUS) with laparoscopy in detecting and characterizing uterosacral ligament (USL) nodules of deep infiltrative endometriosis (DIE) between patients with and without pouch of Douglas (POD) fluid. This prospective study was conducted between June 2021 and July 2023. We included patients from the Second People's Hospital of Shenzhen with two TVUS tests: no POD fluid on the first TVUS test and POD fluid on the second TVUS test. POD fluid was obtained in two ways: naturally occurring fluid during the luteal phase of the menstrual cycle and SonoPODography. Laparoscopic results are the gold standard. To compare the diagnostic performance of TVUS in the diagnosis of DIE on USLs with and without POD fluid. We included 42 patients with a mean age of 36.4 ± 5.4 years. The maximum length diameter for DIE nodules on USLs with and without POD fluid was 13.3 ± 3.3 mm and 10.2 ± 2.5 mm (P < .001), respectively, while the depth of infiltration was 8.1 ± 2.4 mm and 6.1 ± 1.4 mm (P < .001), respectively. When compared to laparoscopic findings, TVUS findings with and without POD fluid resulted in a sensitivity, specificity, accuracy, positive predictive value, negative predictive value area under the curve, and Cohen kappa of 92.3%, 93.8%, 92.9%, 96.0%, 88.2%, 0.930, and 0.850 and 61.5%, 93.8%, 73.8%, 94.1%, 50.0%, 0.766, and 0.499, respectively. Laparoscopy showed USL involvement in up to 59.5% of patients. With or without fluid, the most common ultrasound features of DIE lesions were USL thickening, irregular morphology, clear boundaries, low echo, and no blood flow signal. For patients with clinical suspicion of DIE, TVUS with POD fluid has a higher accuracy for diagnosing USL DIE than TVUS without POD fluid.