Temperature is a critical extrinsic physical parameter that determines cell fate. Hyperthermia therapy has become an efficient treatment for tumor ablation. To understand the response of tumor cells under thermal shocks, we present a paper-based photothermal array that can be conveniently coupled with commercial 96-well cell culture plates. This paper chip device was fabricated in one step using Parafilm® and Kimwipers® based on a heat lamination strategy. Liquid was completely adsorbed and confined within the cellulose fibres of hydrophilic regions. Then, Prussian blue nanoparticles (PB NPs) as the photothermal initiator were introduced into the loading wells, and thermal energy was generated via near infrared (NIR) laser irradiation. After assembling the paper device with a 96-well plate, the temperature of each well could be individually controlled by varying the loading amount of PB NPs and laser irradiation time. As a proof-of-concept study, the effects of local thermal shocks on HeLa cells were investigated using MTT cell viability assay and Live/Dead cell staining. The variation of cell viability could be monitored in situ with controllable temperature elevation. The proposed paper photothermal array loaded with thermal initiators represents an enabling tool for investigating the hyperthermia responses of biological cells. Moreover, the facile fabrication technique for paper patterning is advantageous for customizing high-throughput microfluidic paper-based analytical devices (μPADs) with extremely low cost.