Morphological evolution of layered double hydroxides (LDHs) with preferential crystal facets has appealed gigantic attention of research community. Herein, we prepare hierarchical hybrid material by structurally integrating fusiform-like CuNiAl LDHs petals on conductive backbone of CF (CF@CuNiAl LDHs) and investigate electrocatalytic behavior in nitrate reduction over a potential window of ˗0.7 V to +0.7 V. The CF@CuNiAl LDHs electrode exhibits remarkable electrocatalytic aptitude in nitrate sensing including broad linear ranges of 5 nM to 40 µM and 75 µM to 2.4 mM with lowest detection limit of 0.02 nM (S/N = 3). The sensor shows sensitivity of 830.5 ± 1.84 µA mM1- cm2- and response time within 3 s. Owing to synergistic collaboration of improved electron transfer kinetics, specific fusiform-like morphology, presence of more catalytically active {111} facets and superb catalytic activity of LDHs, CF@CuNiAl LDHs electrode has outperformed as electrochemical sensor. Encouraged from incredible performance, CF@CuNiAl LDHs flexible electrode has been applied in real-time in-vitro detection of nitrite oxidizing bacteria (NOB) through the sensing of nitrate because NOB convert nitrite into nitrate by characteristic metabolic process to obtain their energy. Further, CF@CuNiAl LDHs based sensing podium has also been employed in in-vitro detection of nitrates from mineral water, tap water and Pepsi drink. Display omitted •The hierarchical CF@CuNiAl LDH nanohybrids are synthesized by facile methods.•Maximum interfacial collaborations are resulted from fusiform-like CF@CuNiAl LDH.•CF@CuNiAl LDH hybrids show excellent electrochemical activity for nitrate reduction.•Real-time in-vitro detection of NOB through the sensing of nitrate as biomarker.