The heavy metal pollutions in several environmental-industrial contaminants and living organisms has become a gradually a problem that should not be neglected in all environments. Therefore, this paper aims to improve a novel macrocyclic probe combined with pillar[5]arene and Bodipy for the analysis of Hg (II) in aqueous medium, real sample and living cells. The selectivity and sensitivity of the fluorescent probe towards Hg (II) ions were supported by several spectroscopic parameters such as fluorimetry, absorption, response time, pH effect, competing ion tests and complex stichometry. The limit of detection was determined as 2.18 × 10-7 M. So, the improved macrocyclic and fluorescent innovative sensor has a great potential for selective and sensitive analysis of mercury (II) ion in multi-component metal environments. Moreover, the fluorescent probe showed low toxicity can be used as a reversible and reusable that its sensitivity towards Hg (II) ion was successfully imagined in both living cells and real food samples. The complex stoichiometry between ligand and Hg (II) was determined as 1:2 using Job method. The quenching effect reached to minimum value within about 6 min and the sensor can be used for the detection of Hg (II) in the pH range of 3–9.