Display omitted •Wastewater biofilm formation was monitored by UTDR in-situ and non-destructively.•Biofilm thickness and relatively dense degree could be obtained by UTDR continuously.•3D wavelet analysis visualize the relative biofilm development process over time.•A quick method for estimating bio-carrier applicability for wastewater is presented. Biofilm development plays an important role in facilitating wastewater treatment. The ultrasonic time domain reflectometry (UTDR) is presented for the first time as a tool to assess the initial biofilms development process on bio-carriers. The experiments were carried out with bio samples gathered from practical municipal wastewater and industrial wastewater, respectively. The in-situ and non-invasive UTDR technique was applied to visualize and evaluate the biofilm development process on slices through using the 2D and 3D wavelet analysis by a false color scale. Especially, the distinguished biofilms thickness was observed during the initial biofilm development process via a differential signal, and the relatively dense degree of biofilm were also obtained at the same time. The UTDR measurement could distinguish the initial adherence, reversible adhesion and irreversible adhesion during the initial biofilm formation process. In addition, the UTDR response correlates well with the off-line measurements of the biofilm thickness via an atomic force microscope and optical coherence tomography, suggesting that UTDR-based characterization is a useful tool for in-situ investigating the dynamic processes of predefined biofilm development used for wastewater treatment.