The present study addressed the removal of Trypan blue (TB) from water using a novel Ni-Zn-Bi-layered triple hydroxide (NZB LTH or NZB) synthesized through the co-precipitation technique. The physiochemical properties of NZB were analyzed before and after TB adsorption using XRD, BET, FESEM, FTIR-ATR, Raman, and XPS. Studies on adsorption indicate that 80 mg of NZB has a maximum TB removal effectiveness of around 96.7% at natural pH (~4.5–5.0). This study found that NZB has a maximum adsorption capacity (qmax) of 5.3 mg·g−1 at dye concentrations ranging from 5 to 30 mg·L−1. When combined with various anionic dye mixtures, NZB’s selectivity studies showed that it is highly selective for the removal of TB and is also effective at removing cationic dyes. When compared to Na2SO4 and NaCl salts, NZB had a lower dye removal percentage for TB removal in the presence of Na2SO3. In an adsorption process, the interaction between the TB and NZB in an aqueous solution is caused by hydrogen bonding and electrostatic interactions, which are investigated in the adsorption mechanism. In comparison with ethanol and methanol, the recyclability investigation of NZB revealed the notable removal of TB using 0.1 M NaOH for the desorption. Therefore, the present investigation suggests that NZB is an appropriate adsorbent for the removal of TB from an aqueous solution.