Objective?To evaluate the application of guidewire-assisted biopsy and balloon dilatation cytology smear through ERCP in diagnosis of patients with hilar cholangiocarcinoma.?Methods?During the ERCP procedure, 52 patients with hilar cholangiocarcinoma were treated with balloon dilatation, and cytology smear from the surface of the balloon. At the same time, biopsy forceps assisted by guidewire inserted into the bile duct to biopsy the lesion and obtain specimens.?Results?Success rate of obtaining histopathological through ERCP guidewire assisted biopsy in biliary bile duct was 100.0%, 31 cases were histological diagnosed by forceps biopsy in 52 cases, positive diagnosis rate was 59.6%; The patient with balloon dilatation in 52 cases give cytology smears obtained diagnosis in 22 cases, the positive rate was 42.3%; cytology smear combined with biopsy, a total of 34 cases were diagnosed, the positive rate was 65.4%. There was no serious complications occurred.?Conclusion?Biopsy assisted by guidewire through ERCP is a safe, simple, easy technique and an effective means of obtaining pathological, meanwhile, the cytology smear after balloon dilatation can be used as a supplement to biopsy, and provides an effective means in pathological diagnosis of hilar cholangiocarcinoma.